products & services

EPI GENETICSChIP / Histones / DNA Methylation /Sample Prep / Antibodies / Services

Contents
Company Directory & Ordering Information.................... 1

01

ANTIBODIES
Antibodies for Epigenetics & Gene Regulation
Research............................................................................... 2

02 CHROMATIN ANALYSIS

Chromatin Immunoprecipitation
Overview........................................................................ 4
EpiShear™ Sonication Products.................................... 5
Protein G Beads & Agarose Columns......................... 6
Low Cell ChIP-Seq Kit.................................................... 7
ChIP-IT High Sensitivity® ................................................ 8
ChIP-IT® Express.............................................................. 9
TAG - ChIP-IT®............................................................... 10
enChIP.......................................................................... 11
ChIP-IT® ChIP-Seq........................................................ 12
ChIP Kits for Disease Research.................................. 13
Next Gen DNA Library Kit........................................... 16
ChIP Normalization..................................................... 17
Chromatin Assembly
Chromatin Assembly Kit............................................. 18
NOMe-Seq................................................................... 19
Cell Free DNA Purification Kit..................................... 20
NU.QTM Total.................................................................. 21

DNA METHYLATION
Overview............................................................................. 22
Reagents for DNA Methylation Analysis
Antibodies.................................................................... 23
DNA Methylation Enzymes......................................... 24

03

Global DNA Methylation Analysis
Global DNA Methylation Assay – LINE-1................... 25
Global 5-hmC Quantification Kit............................... 26
Targeted DNA Methylation Analysis
MeDIP........................................................................... 27
MethylCollector™ Ultra............................................... 28
hMeDIP......................................................................... 29
Hydroxymethyl Collector™......................................... 30
Bisulfite Conversion Kit................................................ 31
ChIP-Bis-Seq................................................................. 32
DNMT Activity / Inhibition Assay....................................... 33
Methylated DNA Standard Kits................................. 34

04 HISTONE ANALYSIS

Overview............................................................................. 35
Histone Peptide Array
MODified™ Histone Peptide Array............................. 36
MODified™ Array Labeling Kit.................................... 36
MODified™ Protein Domain Binding Kit..................... 37

Histone Purification Kits...................................................... 38
Histone Acetyltransferase (HAT) Assays........................... 40
Histone Deacetylase (HDAC) Assays............................... 40
Histone Demethylase (HDM) Assays................................ 41
Histone Quantification ELISAsHistone Modification ELISAs....................................... 42
Total Histone H3 ELISA................................................. 42
Histone H3 PTM Multiplex Assay........................................ 43

05 REAGENTS FOR DRUG DISCOVERY

Recombinant Proteins & Enzymes............................ 44
Recombinant Histones & Nucleosomes................... 44
Antibodies.................................................................... 44
Small Molecules.......................................................... 44
Acetyltransferases & Deacetylases.......................... 44
Methyltransferases & Demethylases......................... 44
Reader Domains......................................................... 44
Kits & Reagents for Drug Discovery........................... 45

06 NON-CODING RNASLightSwitch™ Luciferase Assay System Overview............ 46
LightSwitch™ 3´UTR and miRNA Products................. 46
LightSwitch™ lncRNA Promoter Products.................. 46

miRNA Target IP Kit............................................................. 48
RNA Subcellular Isolation Kit.............................................. 49

07 SERVICES

Epigenetic Services............................................................ 50
ChIP-Seq and ChIP-qPCR Services........................... 51
Super-Enhancer Profiling Service.............................. 52
ChIP-Seq Spike-in Normalization Service................. 53
ChIP Antibody Validation Service............................. 55
RNA-Seq Service......................................................... 56
Pol II ChIP-Seq Service................................................ 56
Reduced Representation Bisulfite Sequencing....... 57
DNA Methylation Analysis Services........................... 58
Mod Spec®................................................................... 59
Histone PTM Quantitation Service............................. 60
Interactome Profiling (RIME) Service........................ 61
Bioinformatic Solutions............................................... 62

Gene Regulation & Functional Genomics Services....... 63
Custom Cloning & Mutagenesis Services................ 63
Sequence Variant Assay Services............................. 63
LightSwitch™ System Applications............................ 64

Active Motif is the industry leader in providing innovative tools to enable
epigenetics and gene regulation research. We provide superior products,
service and support to serve our life science, clinical, pharmaceutical and drug
discovery partners. We are recognized worldwide for our ground-breaking ChIP
technologies, for our LightSwitch™ functional genomic products and genomewide services. Whether you are an expert in the field of epigenetics or gene
regulation or a researcher looking to integrate these areas of research into yourstudies, our comprehensive portfolio of products offers end-to-end solutions tomeet the specific needs of your research.

ACTIVE MOTIF, INC.
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008 USA
For distributors, visit
www.activemotif.com/distributors

WEB:
www.activemotif.com

SALES:
sales@activemotif.com

US: 1 877 222 9543
EUROPE: +32 (0)2 653 0001

CUSTOMER SERVICE:
orders@activemotif.com
TECHNICAL SUPPORT:
tech_service@activemotif.com

JAPAN: +81 (0)3 5225 3638
CHINA: 400 018 8123

1

Antibodies

01

ANTIBODIES FOR EPIGENETICS & GENE REGULATION RESEARCHANTIBODY PORTFOLIO
Active Motif offers a wide range of antibodies
specific for epigenetic modifications, chromatin
modifying proteins, and transcription factors.
From immunogen design to specificity screening
and technique validation, our staff scientists
have an established program to qualify ourantibodies for use in various applications,
including ChIP-Seq, EMSA, Western blot andimmunofluorescence. Our years of experiencein antibody development ensures that only thehighest quality antibodies are offered for use inyour research.

AbFlex®Recombinant
Monoclonal
Polyclonal
Epitope Tag
ChIP Validated
ChIP-Seq Validated

WE HAVE WHAT YOU WANT
The antibodies list below shows only a small sampling of the over 800 antibodies currently offered.
For a complete, up-to-date list of all available antibodies, please visit www.activemotif.com/abs.
HISTONES & HISTONE MODIFICATIONSDNA METHYLATION

H1

H3K27me1

3-mC

DNMT3ASTEM CELLS
BMI-1

Notch1

H2A

H3K27me2

5-caC

DNMT3B

c-Myc

Notch3

H2A.X

H3K27me3

5-fC

DNMT3L

EED

YY1

H2A.Z

H3S28ph

5-hmC

TETs

EZH2

Oct4

H2B

H3K36ac

5-mC

MBDs

KLF4

Ring1B

H3

H3K36me2

DNMT1

MeCP2

LIN28A

Sox2

H3K4me1

H3K36me3

DNMT2

Uhrf1

Nanog

Sp1

H3K4me2

H3K56ac

NKX2.5Suz12

H3K4me3

H3K79me1

CHROMATIN MODIFIERS

H3K9ac

H3K79me2

BRD3

Jhd2

GENE REGULATION

H3K9me1

H4

BRD4

JMJD2A & 2D

Ago1/2/3

IKK

H3K9me2H4ac (pan-acetyl)

CARM1

EHMT2

AML

MyoD

H3K9me3

H4R3me2a

CGBP

LSD1 / KDM1A

AP-1

NFκB

H3S10ph

H4K5ac

DOT1L

MLL / HRX

AR1

p53

H3T11ph

H4K12ac

EZH1& 2MMSET / WHSC1

Cas9

PARP

H3K14ac

H4K16ac

GCN5

PARPs

Dicer

PP2A

H3K18ac

H4K20me1

HDACs

PRMTs

ER1

RNA pol II

H3K27ac

H4K20me2

HP1

Sirtuins

FKHR

STAT

H3R17me2aH4K31me1

JARID1C / KDM5C

SUV39H1IκBαUbiquitin

2

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01

Antibodies

COMPETITIVE AB-VANTAGE
Not every antibody is suitable for ChIP
One of the challenges for epigenetics research is the lack of antibodies that have beenvalidated for use in techniques such as ChIP and ChIP-Seq. The problem is compoundedby antibody suppliers who do not manufacture or test the antibodies they sell, and who sellthem to one another and then to researchers. At Active Motif, we manufacture and rigorously
test our antibodies in-house to ensure their quality and performance. Our ChIP validated
antibodies (Figure 1) are validated according to the guidelines of the ENCODE Consortium*
and are run through the same internal validation program used by our ChIP-Seq Services group.
In addition, we are the only company to test the specificity of our histone antibodies using
our ground-breaking MODified™ Histone Peptide Array (Figure 2, page 36) that enables
assessment of cross-reactivity of our antibodies against a panel of known histone modifications
in a single experiment. Our array offers the most extensive coverage available for commercial
arrays of similar format and enables the study of not only individual sites, but also the effects
of neighboring modifications on recognition and binding.

Control

*

140

*

*

120

EstrogenSpecificity Factor

SRC3
ChIP-Seq*

Control
Pol IIChIP-Seq
Estrogen

Histone H3K9me3 pAb

100
80
60
40
20

RET
q11.2142,880,000

0H342,900,000

42,920,000

42,940,000

K9

H3

me

3

K2

H3

7m

e3

R2

H3

me

2s

R2

H3

me

2a

K4

ac

H3

K4

H3

me

2

R8

H3

me

2s

K4

H3

me

3

K4

H3

me

1

R2

me

2a

F IG U R E 1 : Active Motif’s ChIP validated antibodies are qualified using
the same internal validation program used by our Custom Services group.

F IGU RE 2:  Active Motif’s MODified Histone Peptide Arrayconfirms the specificity of modification-specific histone antibodies.

ChIP-Seq data from control and estrogen-treated MCF-7 chromatin using
antibodies against RNA pol II CTD phospho Ser2 (Catalog No. 61083) andagainst the estrogen-inducible transcription factor SRC3 (Catalog No. 39797).

Data produced using the MODified Histone Peptide Array to confirm
the specificity of Histone H3K9me3 antibody (Catalog No. 39161).

VISIT OUR WEBSITE
Active Motif is continually adding new antibodies to its portfolio. For a complete,
up-to-date list of available antibodies, please visit us at www.activemotif.com/abs.
*For ENCODE Consortium guidelines for antibody validation, please refer to Landt S.G. et al. (2012) Genome Res. 22, 1813–1831.

www.activemotif.com

3Chromatin Analysis

02

CHROMATIN IMMUNOPRECIPITATION
Active Motif offers a complete selection of validated reagents, instruments and kits to help
streamline each step of the ChIP assay workflow. Whether you are performing ChIP for the
first time or are an expert in need of a specialized ChIP protocol and reagents, Active Motif
has the right products to meet your needs. For more complete information on available
ChIP products, please visit us at www.activemotif.com/chip.

EPISHEAR™
SONICATORS
Probe sonicators
and accessories forshearing chromatinin preparation for
ChIP analysis.

ChIP-IT® ChIP KITS
ChIP-IT® offers a comprehensive
suite of basic and specialized ChIP
kits for performing standard chromatin
immunoprecipitation or more innovativetechniques, such as sonication-free ChIP,
ChIP without the need for protein-specificantibodies, sequential ChIP, transcription
factor ChIP, low-cell ChIP, RNA-ChIP, highresolution ChIP and various other methods.
For optimal results & the most innovative
ChIP technologies, rely on the
ChIP Experts™.

ChIP
ACCESSORIES
A variety of ChIP accessories,
including ChIP DNA purification
kits, Protein G magnetic beads
and agarose columns, qPCR
primer sets and controls, tocomplement your ChIP
experiments.

4

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02

Chromatin Analysis

EPISHEAR™ SONICATION PRODUCTS
Active Motif’s EpiShear™ sonication systems are designed for ease of use and ultimate
control for the highest degree of sample-to-sample reproducibility. Keypad programming anddigital displays clearly display all parameters and options, making it easy to control functions
such as amplitude, pulse cycles and processing times with the simple touch of a button.

Experience the versatility of probe sonication
The EpiShear™ Probe Sonicator is ideal for shearing chromatin, DNA
and RNA, or for cell disruption and homogenization. With a varietyof microtip probe sizes available, you canprocess samples ranging from 200 µl to 50 ml.
Each unit includes:

TECHNICAL SPECS
Input Voltage:Cat. No. 53051: 100V - 120V @ 50/60 Hz
Cat. No. 53052: 220V - 240V @ 50/60 Hz
Adjustable Amplitude: 20% - 100%Programmable Timer: 1 sec - 10 hrs

•
•
•
•
•

an ultrasonic electric generator
a piezoelectric converter
a 1/8” microtip probe
power & converter cables
a wrench set for changing probes

Generator Dimensions:8.0" W x 13.75" L x 5.75" H
7.0 lbs.
(203 mm x 349 mm x 146 mm)(2.2 Kg)
EpiShear Probe Sonicator

The unit is backed by a two-year warranty.
For more information, visit www.activemotif.com/probe.

Sample size (probe dependent):
200 µl - 50 ml(500 µl - 15 ml with included 1/8” probe)
Adjustable Pulse: 1 - 59 seconds
Frequency: 20 kHz; Power: 120 watts

Digital
Display
Bar Graphof % Power
Start/Stop
Sonication

Power
On
Set
Timer
Set Pulse
On/Off Times

500bp

AdjustAmplitude

Set
Amplitude

EpiShear™ Probe Sonicator

www.activemotif.com

100bp
1

2

3

4

F IGU RE: Gel analysis of sheared HeLa chromatin.

ORDERING INFORMATION
Product

1000bpPower OffFormatCat. No.

110V

53051

230V

53052

Three samples of chromatin were prepared fromHeLa cells for ChIP analysis using the EpiShear Probe
Sonicator (1/8” probe) with the ChIP-IT® Express Kit(Catalog No. 53008) components and protocol using
5, 10 and 20 pulses at 25% amplitude. Each pulse
consisted of a 20-second sonication followed by a30-second rest on ice to prevent heat build up. The
samples were then run on a 1% agarose gel.

5

Chromatin Analysis

02

PROTEIN G BEADS & READY-TO-ChIP AGAROSE COLUMNS

For researchers who routinely perform ChIP with their own optimized protocols, Active Motif
is making it faster & easier to obtain consistency in chromatin preparation and improve thequality of your ChIP reactions with the use of our low background Protein G Magnetic Beads
or Agarose Columns and our ChIP-IT® Fixation Buffer.

Magnetic pull-down for faster, more efficient processing
Active Motif offers the ChIP-IT® Protein G Magnetic Beads, the same Protein G magnetic beads that are usedin our best-selling ChIP-IT® Express Kits (page 8) to streamline the protocol and enable ChIP to be performedin just 1 day. Because the beads are pre-blocked and there is no resin loss, the use of magnetic beads allows
for rapid processing and improved efficiency of your ChIP procedure, giving you the advantage of:

✓
✓
✓
✓
✓

Fewer steps
Minimal sample loss
High specificity
Less background
Validated use for various applications including ChIP & Co-IP

Improve the reproducibility of your optimized ChIP protocol
Active Motif’s Protein G Agarose Columns contain the same high-affinity Protein G agarose
beads used in our highly popular ChIP-IT High Sensitivity® Kit (page 12). The beads havebeen specifically engineered to bind IgG with high affinity and to eliminate non-specificbinding. The beads offer many advantages for ChIP over traditional agarose beads including:Protein G Agarose Column

✓
✓
✓
✓
✓

High binding capacity: 10 µg IgG / µl bead
Better reproducibility
No sample loss
Filtration method is faster & easier than centrifugation
Adaptable to various applications

ORDERING INFORMATION
Product

FormatProtein G Agarose Columns
ChIP-IT® Protein G Magnetic Beads
ChIP-IT® Fixation Buffer
6

ASIA: +81 (0)3 5225 3638

30 rxns
5 rxns
40 rxns (1 ml)
200 rxns (5 ml)
3 ml
EUROPE: +32 (0)2 653 0001

Cat. No.
53039
53037
53033
53034
53038
NORTH AMERICA: 877 222 9543

02

Chromatin Analysis
LOW CELL CHIP-SEQ KIT

Complete ChIP-Seq workflow from limited cell numbers
Active Motif’s Low Cell ChIP-Seq Kit provides a complete ChIP-Seq workflow for chromatin preparation,
immunoprecipitation, and next generation sequencing library preparation from limited amounts of cells or
small tissue biopsies. The Low Cell ChIP-Seq Kit not only reduces sample input requirements, but it also resolves
issues often associated with low cell ChIP, including poor signal-to-noise ratios, inefficient library amplification,and high duplication rates.

✓ Reproducible ChIP-Seq data from as little as 1,000 to 50,000 cells, depending on protein abundance✓ Includes reagents for chromatin preparation, immunoprecipitation and the Next Gen DNA Library Kit(page 16)

✓ Maximize unique data by using Molecular Identifiers (MIDs)✓ Better peak calling from improved signal-to-noise ratios

F IGU RE 1: Low Cell ChIP-Seq peaks
compared to ENCODE data sets.
ChIP-Seq data shows peaks from BRD4
(10,000 cells), H3K27ac (5,000 cells) andH3K27me3 (5,000 cells) are high quality andcomparable to ENCODE data sets using 20
million cells. Libraries were constructed with
the Next Gen DNA Library Kit (page 16)
reagents included in teh Low Cell ChIP-Seq
Kit.

F I G U R E 2 : MID-containing Low Cell ChIP-Seq Library Molecules.
ChIP-Seq libraries generated with the Low Cell ChIP-Seq Kit containsample indices (indicated in dark blue) for sample multiplexing during
sequencing, and MIDs (indicated in teal) for identification of uniquelibrary molecules. MIDs enable users to save more of their sequencing
data by retaining molecules that would otherwise be recognized as
duplicates and be discarded.

ORDERING INFORMATION
Product

FormatCat. No.

Low Cell ChIP-Seq Kit
ChIP Buffer
Blocking Reagent AM1
BSA (10 mg/ml)
BlockerProtein G Agarose Beads

16 rxns
50 ml
0.1 ml0.1 ml0.1 ml1.2 ml

53084
37516
37496
37497
37498
37499

TE, pH 8.0

35 ml

37515

www.activemotif.com

7

Chromatin Analysis

02

ChIP-IT HIGH SENSITIVITY®
Most sensitive transcription factor ChIP Kit available
The ChIP-IT High Sensitivity® Kit’s specially formulated buffers enable highly
efficient enrichment from low abundance targets, such as transcription factors,
or from limited sample amounts or low affinity antibodies. During theimmunoprecipitation reaction, low-background Protein G agarose beads
and an antibody blocker reduce non-specific binding. Specialized ChIP
buffers are designed to enhance enrichment and reduce the presenceof non-specific DNA. In addition, ChIP filtration columns are used for faster,easier and more consistent capture and wash steps. The result is a kit that is
optimized to deliver both higher signals and lower background whencompared to other commercially available ChIP kits (Figure 1).

✓ Perform ChIP reactions on low abundance transcription factors (TFs),
with limited sample material or using low-affinity antibodies (Figure 2)✓ Enrich DNA from as little as 1,000 cells per IP reaction✓ Validated across multiple sample types✓ Proven performance in ChIP-Seq, ChIP-chip and qPCRTECHNICAL SPECS
Recommended for use with:- low abundance targets (e.g. TFs)
- limited sample material- low affinity antibodies
Cell requirements (# cells / ChIP rxn):- high abundance target: ≥ 1K cells- low abundance target: ≥ 50K cells
Recommended # cells forchromatin preparation: 100K cells
Capture method: Column filtration
IP method: Protein G agarose beads
Procedure length: 2 - 3 days
Compatible downstream applications:- qPCR- ChIP-chip- ChIP-Seq

For more information, visit www.activemotif.com/chipiths.

Binding events detected / 1,000 cells

ChIP Kits Targeting Low Abundance Transcription Factors
30

20

NCOA2 antibody

High background

Neg Ctrl IgG

10

No enrichment
Non-specific binding0

Neg Ctrl
primers

ESRRA
primers

ChIP-IT® HS

Neg Ctrl
primers

ESRRA
primers

Competitor M

Neg Ctrl
primers

ESRRA
primers

Neg Ctrl
primers

Competitor I

ESRRA
primers

Competitor D

F IG U R E 1 : ChIP-IT High Sensitivity shows better enrichment thancompetitor ChIP Kits.

F IGU RE 2: ChIP-Seq data showing peaks from transcriptional
co-factor NCOA2-enriched DNA generated using ChIP-IT High Sensitivity.

A comparison of ChIP kits targeting a low abundance transcription factor
was performed using an antibody for the low abundance NCOA2 protein
and a negative control IgG. Following enrichment, qPCR was performed
using the ChIP-IT® qPCR Analysis Kit (Catalog No. 53029) in order to allow
stringent normalization of the data for direct comparison of the results.

ChIP-Seq data showing peaks from transcriptional cofactor NCOA2 in
the promoters of two genes. ChIP was performed using the ChIP-IT HighSensitivity Kit, confirmed with the ChIP-IT qPCR Analysis Kit and followed bysequencing on the Illumina Next-generation sequencing platform.

ORDERING INFORMATION
Product
ChIP-IT High Sensitivity Kit
High Sensitivity Chromatin Preparation Kit
ChIP-IT® qPCR Analysis Kit®

8

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FormatCat. No.

16 rxns
16 rxns
10 rxns

53040
53046
53029

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NORTH AMERICA: 877 222 9543

02

Chromatin Analysis
ChIP-IT® EXPRESS

Single day optimized ChIP with ChIP-IT® Express
Active Motif’s ChIP-IT Express Kits use ChIP-IT® Protein G Magnetic Beads (page 7) that
reduce processing time with fewer steps and reagents, making it possible to perform
ChIP in just 1 day (Figure 1, opposite page). Kits that use either sonication or enzymatic
chromatin shearing protocols are available. Both kits offer the following advantages:

✓
✓
✓
✓

Complete Solution – includes all necessary components for performing ChIP
Results in just 1 day – 4-hour IP, easy-to-follow streamlined protocol
Convenient – procedure is already optimized for superior results
Sensitive – as few as 100,000 cells needed per ChIP reaction
ChIP-IT Express ChIP Kit

For complete details, please visit www.activemotif.com/chipitexpress.

A

F IGU RE 1: Comparisonof DNA fragmentation resultsfrom the ChIP-IT Express
Sonication and Enzymatic
shearing procedures.

B

1000 bp

Sheared chromatin preparedusing either (A) the ChIP-IT
Express Shearing Kit and the
EpiShear™ Probe Sonicator or(B) the ChIP-IT Express Enzymatic Shearing Kit.

1000 bp

500 bp500 bp100 bp100 bp
1

2

3

1

4

2

3

4

5

Binding events detected per 1000 cells

600

F IGU RE 2: H3K4me3
enrichment at GAPDH
promoters, but not genedeserts.

500

400

300

200

100

0

Negative Primer
Set 1

Negative
Gene Desert

Positive Primer Set
GAPDH-2

The ChIP-IT Express Kit wasused to perform ChIP using
MCF-7 chromatin and Histone
H3K4me3 antibody qPCR
analysis of enriched DNA
using Negative and Positive
Control Primers, and primers
specific to a gene desert
show enrichment of H3K4me3
at GAPDH promoters, but not
at gene deserts, as expected.

TECHNICAL SPECS
Available versions*:- Enzymatic (ChIP-IT® Express Enzymatic)- Sonication (ChIP-IT® Express)
Recommended for use with:- high abundance proteins- ChIP validated antibodies
Sufficient reagents for:- 10 chromatin preparations- 2 shearing optimizations- 25 ChIP reactions
Cell requirements: ≥100K cells / ChIP rxn
Capture method: Magnetic
IP method: Protein G Magnetic Beads
Procedure length: 4 hours*Protein G Magnetic Beads are also
sold separately (see Ordering Information on page 6).

ORDERING INFORMATION
Product

FormatCat. No.

ChIP-IT Express Kit
ChIP-IT® Express Enzymatic Kit

25 rxns
25 rxns

53008
53009

®

www.activemotif.com

9Chromatin Analysis

02

TAG-ChIP-IT®
Perform ChIP without protein-specific antibodies
Active Motif’s Tag-ChIP-IT® Kit addresses a commonly encountered issue when considering ChIP, the lack ofavailable ChIP-grade antibodies to a target of interest. A common solution to this issue is to use an epitopetag, such as FLAG, HA, and GFP, to tag the protein of interest. However, since these tags are not specificallydesigned for ChIP, they have a high and unpredictable failure rate. Tag-ChIP-IT is the first epitope-tagging
procedure that is specifically optimized for ChIP.

How does it work?
Tag-ChIP-IT utilizes a unique AM-tag (patent pending) designed specifically for ChIP consisting of a small,unstructured sequence that can be attached to any protein of interest. Furthermore, we have designed ahigh specificity antibody to the tag for maximum pull-down efficiency during immunoprecipitation (IP).
Combined with optimized reagents and a streamlined protocol, this unique ChIP method enables researchers
the ability to perform ChIP on any target without the need for target-specific antibodies. For more complete
information, please visit us at www.activemotif.com/tagchip.

✓
✓
✓
✓

Ideal for targets lacking ChIP-qualified antibodies
Improved sensitivity & specificity over FLAG, GFP and HA tags
Negligible cross-reactivity with mammalian systems
Study isoforms, mutations & truncations with ease

Input

ER Motif Identified

Endogenous ER
AM-tag ER#1

SampleAM-tag ER#1

AM-tag ER#2

AM-tag ER#2

F IG U R E :   Tag-ChIP-IT identifies same estrogen receptor (ER) binding motifs as published ChIP-Seq data.
ER cDNA was cloned into pAM_1C Empty Vector and transiently transfected into cells. Cells were
induced with estradiol and ChIP was performed on harvested chromatin using the Tag-ChIP-IT Kit.
Enriched DNA was submitted for Next-generation sequencing. Data was compared to published
ChIP-Seq results from ER antibody enrichment performed under the same conditions. ChIP-Seq datashows the same ER peak profile with the AM-tag ChIP as endogenous ER. Detected binding sites were
further evaluated for binding motifs. Results show the ER motif was identified in both Tag-ChIP-IT samples.

WHAT’S IN THE BOX?
The Tag-ChIP-IT Kit contains all
the buffers and reagentsnecessary for ChIP analysis
using the AM-tag. The pAM_1C
Empty Vector is available
separately and is used toclone your protein of interest
in-frame with the C-terminal
AM-tag. Alternatively, the AMtag sequence can be cloned
into your expression vector of
choice. Following transfectionand expression of your tagged
protein, the Tag-ChIP-IT Kit
is used to isolate chromatin
and perform IPs using AM-Tag
antibody specific for the AM-tag.

ORDERING INFORMATION
Product

FormatCat. No.

Tag-ChIP-IT® Kit
pAM_1C Empty Vector

16 rxns
20 µg
0.2 ml
0.5 ml

53022
53023
32042
32043

FuGENE® HD Transfection Reagent
10

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NORTH AMERICA: 877 222 9543

02

Chromatin Analysis
enChIP

Determine CRISPR/Cas9 specificity with enChIP
To enable you to assess Cas9 specificity, Active Motif’s enChIP Kit (Engineered DNA-binding molecule-mediatedChromatin Immunoprecipitation) offers a modified ChIP assay that utilizes the CRISPR/Cas9 system to target a
specific DNA locus for immunoprecipitation (Figure 1). Using the enChIP Kit for your genome editing experiments
enables you to biologically validate each target sequence for specificity. Sequences demonstrating off-target
effects can be excluded, saving valuable time and resources.

✓
✓
✓
✓

Identify off-target binding events for each gRNA sequence

dCas9
DNA loop

Evaluate cis- and trans-interacting chromosomal looping events

AM-tag
gRNA

ChIP-optimized AM-tag design ensures highly efficient dCas9 enrichment

RNA
proteinWorks well with open chromatin regions to detect promoter, enhancer
and insulator elements

F IGU RE 1: Illustration of enChIP design.

How does it work?
A guide RNA (gRNA) containing 20 nucleotides complementary to the desired genomic region is expressed in
combination with an enzymatically inactive Cas9 protein (dCas9) tagged with an AM-tag that is specificallydesigned for ChIP. The gRNA directs dCas9 to the target sequence and an RNA-DNA heteroduplex is formed.
Chromatin immunoprecipitation is then performed using an antibody directed against the dCas9 AM-tag toenrich for genomic sequences bound by the gRNA/dCas9 complex. By using the enChIP Kit, off-target gRNA bindingsites can be identified (Figure 2). This provides valuable information regarding the quality of gRNA design prior touse in genome editing experiments. Additionally, the enChIP Kit can be used to study cis- and trans-interacting
chromosomal looping events. For more complete information, please visit us at www.activemotif.com/enchip.
CTCF

Target Site

ChIP-Seq

gRNA2

gRNA1

No gRNAF IGU RE 2:  enChIP validates specificity of gRNA for a CTCF binding site.
Two gRNAs were designed targeting different 20 nt sequences within a 500 bp
region of a CTCF binding site on chromosome 19. Each gRNA sequence
was cloned into the pAM_dCas9 vector and transfected into HEK293T cells.
A ‘No gRNA’ negative control was also performed. Chromatin was preparedand immunoprecipitated according to the enChIP Kit. Enriched DNA was
analyzed by Next-generation sequencing and background was subtractedusing the ‘No gRNA’ control. Data was compared to ChIP-Seq data for CTCF
in the same cell line. Results show gRNA1 had a strong peak and specificbinding at the target location. Data for gRNA2 revealed a large number ofoff-target binding events outside of the target location. This confirms that
enChIP provides biological validation for CRISPR/Cas9 gRNA specificity.

ORDERING INFORMATION
Product

FormatCat. No.

enChIP Kit
pAM_gRNA Vector
pAM_dCas9 Vector
pAM_gRNA_CTCF Vector (Positive control)
pAM_dCas9_CTCF Vector (Positive control)

16 rxns
10 µg
10 µg
10 µg
10 µg

53125
53121
53122
53123
53124

www.activemotif.com

11

Chromatin Analysis

02

ChIP-IT® ChIP-SEQ
ChIP for Next-generation sequencing
The combination of ChIP with genome-wide analysis using Next-generation
sequencing (ChIP-Seq) is a powerful approach that can provide insights
into gene regulation, the impact of chromatin modifications on gene
expression and signaling pathway mechanisms. Active Motif’s ChIP-IT®
ChIP-Seq Kit is designed to provide the highest quality ChIP-enriched DNA
for use in sequencing on Illumina® Genome Analyzer II, HiSeq and MiSeq
Next-generation sequencing systems.

TECHNICAL SPECS
Recommended for use with:- low abundance targets (e.g. TFs)
- low affinity antibodies
Sufficient reagents for:- 16 chromatin preparations- 16 chromatin IPs- 10 sequencing libraries
Cell requirements: ≥ 1.5M cells / ChIP rxn

ChIP-IT ChIP-Seq is robust enough for use with challenging antibodies
that do not yield signal with other ChIP methods and sensitive enoughto detect binding of low abundance transcription factors. The increased
sensitivity is due to optimized ChIP buffers which reduce the presence of
non-specific DNA, resulting in lower background and better enrichment.
Better enrichment translates into larger ChIP-Seq peaks and more accurate
peak calling, since a greater percentage of the sequence reads are
mapped to binding sites rather than background DNA regions. To learn
more, please visit us at www.activemotif.com/chipitseq.

Recommended # cells forchromatin preparation: ≥ 4.5M cells
Capture method: Column filtration
IP method: Protein G agarose beads
Procedure length: 2 - 3 days
Compatible downstream applications:- qPCR- ChIP-chip- ChIP-Seq

WHAT’S IN THE BOX?
The ChIP-IT ChIP-Seq Kit contains the same robust
chromatin IP components of the ChIP-IT High Sensitivity®
Kit (see opposite page) along with other components
necessary for preparation of samples for ChIP-Seq
analysis. Included in the ChIP-IT ChIP-Seq Kit are:•

Optimized protocol & buffers to perform ChIP
from fresh or frozen cells or tissues•

DNA purification reagents for clean-up of
ChIP-enriched DNA

F IG U R E : ChIP-IT ChIP-Seq reveals that H3K4me3 peaks arepresent at the start site of all Zfp genes.

•

ChIP-IT ChIP-Seq was performed using a Histone H3K4me3 pAb(Catalog No. 39159) on mouse livers. The data shows the presenceof H3K4me3 at the transcription start sites of several Zfp genes.

ChIP-IT® qPCR Analysis Kit for the verification ofquality of the DNA prior to sequencing•

Library construction reagents and protocol forthe preparation of 10 NGS libraries

ORDERING INFORMATION
Product

FormatCat. No.

ChIP-IT® ChIP-Seq Kit

10 libraries

53041

12

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02

Chromatin Analysis
ChIP KITS FOR DISEASE RESEARCH

For disease research, Active Motif provides the ChIP-IT® FFPE and ChIP-IT® PBMC assays to
finally enable researchers to perform ChIP analysis of primary tissue samples. Performing ChIPon these sample types had been previously unsuccessful due to the difficulty in extracting
high-quality chromatin from difficult-to-lyse, very limited, or degraded material.

ChIP-IT® FFPE for ChIP analysis of FFPE archived tissue samples
Active Motif’s ChIP-IT FFPE assay is the first-of-its-kind to enable extraction and ChIP
analysis of chromatin from FFPE samples for Next-generation sequencing, enabling
researchers to uncover epigenetic variances in archived tissue. The assay comes in
two modules:•

ChIP-IT® FFPE Chromatin Preparation Kit – extract high-quality ChIP-grade chromatin from preserved
human, mouse or rat FFPE material. The kit has been successfully used to extract chromatin from FFPE blocks
stored over 10 years under suboptimal conditions.

•

ChIP-IT® FFPE Kit – perform ChIP using the only ChIP kit available that can enrich for ChIP DNA using FFPEextracted chromatin. The assay produces ChIP-enriched DNA in sufficient amounts and quality for analysis
by qPCR or Next-generation sequencing.

ChIP-IT® PBMC for ChIP analysis ofdifficult-to-lyse T and B cells

H3K4me1

Active Motif’s ChIP-IT PBMC Kit is the only ChIP kit available
to enable ChIP on difficult-to-lyse peripheral blood mononuclear cells, or PBMCs, including lymphocytes (T cells, B cells& NK cells) and monocytes. PBMCs are highly resistant tolysis under conditions normally suitable for other cell types.
We have developed an optimized chromatin extraction
method that yields high-quality chromatin from primary cells
for ChIP analysis and Next-generation sequencing. ChIP
reagents have also been optimized to enable enrichment
of DNA from nanogram quantities of chromatin and still
produce robust signal and minimal background.

H3K27ac*
WT

H3K27me3

DKO

ORDERING INFORMATIONF IGU RE: ChIP performed with chromatin from primarymouse T cells using the ChIP-IT PBMC Kit.
Epigenetic profiling was performed in a mouse model harboringa knockout (DKO) of a lymphoma related gene. Within the regionof the genome shown, there is a clear loss of the enhancer
associated H3K27ac modification at a location upstream of the
Cfc1 gene (red asterisk*).

Product

FormatCat. No.

ChIP-IT® FFPE Chromatin Preparation Kit
ChIP-IT® FFPE Kit
ChIP-IT® PBMC Kit

5 rxns
16 rxns
16 rxns

53030
53045
53042

www.activemotif.com

13

Chromatin Analysis

02

SPECIALIZED ChIP KITS & ACCESSORIES
Active Motif includes a variety of ChIP Kits for specialized applications along with ChIP
accessory products in our vast portfolio of ChIP-related products and services. As the
ChIP Experts™, we are continually striving to develop and provide new and innovative
ChIP products and services to meet our customer’s needs.

Specialized ChIP Kits
• Re-ChIP-IT® – perform sequential ChIP to simultaneously assay two unique binding eventsat the same genomic locus.
• RNA ChIP-IT® – recover immunoprecipitated RNA for RT-PCR analysis to study RNA-protein
interactions in a chromatin context.
• ChIP-Bis-Seq (ChIP-Bisulfite-Sequencing) – provides single nucleotide resolution DNA
methylation profiling by combining ChIP with bisulfite conversion and sequencing (Bis-Seq).

ChIP Accessory Products• ChIP Control qPCR Primer Sets – large variety of qPCR primer sets for use as positive andnegative controls for the more common ChIP targets. For a complete list of available
products and ordering information, please visit www.activemotif.com/qpcr.
• ChIP-IT® Control Kits – positive and negative control antibodies, a Bridging Antibody anda positive control PCR primer set to assess your ChIP procedure by endpoint PCR.
• ChIP-IT® Control qPCR Kits – positive control RNA pol II antibody, a Bridging Antibody toenhance the binding affinity of mouse monoclonal antibodies, a negative control IgG
antibody, and species-specific positive and negative control qPCR primers.
• ChIP-IT® qPCR Analysis Kit – includes a standard curve DNA and primer pair, human andmouse positive and negative control qPCR primer sets and an analysis spreadsheet tosimplify qPCR data analysis and assess the quality of ChIP-enriched DNA.
• Ready-to-ChIP Chromatin – offered from a number of ENCODE cell lines that have been
optimally sheared by sonication and validated in ChIP as a control or test sample.
• Bridging Antibody – to enhance the binding affinity of mouse monoclonal antibodies.
• Dounce Homogenizer – available in two sizes for preparation of cell lysates or chromatin.

14

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02

Chromatin Analysis

ChIP DNA Purification Kit
Active Motif’s ChIP DNA Purification Kit is specificallyformulated to remove common contaminants, such as
reverse cross-linking reagents, Proteinase K or other digestive
enzymes, detergents and salts from eluted ChIP DNA samples.
The kit enables efficient and rapid preparation of ChIP DNA
for analysis without the need for messy, labor intensive andtime-consuming phenol/chloroform extraction. Our buffersand columns are specifically optimized for purification of
ChIP DNA to produce the highest quality DNA for use indownstream chromatin IP and epigenetics applications.

Correct pH
pH < 7.5

Wrong pH
pH > 8.5

Wrong pH
pH > 7.5

F IGU RE:  The DNA Purification Binding
Buffer has a pH indicator dye so the pH of thesolution can be easily determined.

✓ Specifically formulated for ChIP DNA clean-up✓ 85-100% recovery of purified DNA✓ Start with as few as 10,000 cells✓ Enables recovery of DNA fragments as small as 50 bp✓ Proven compatibility with ChIP and methylated DNA enrichment kits & protocols

ORDERING INFORMATION
Product

FormatCat. No.

Re-ChIP-IT® Kit
RNA ChIP-IT® Kit
RNA ChIP-IT® Control Kit – Human
ChIP-Bis-Seq Kit
ChIP-IT® Control Kit – Human
ChIP-IT® Control Kit – Mouse
ChIP-IT® Control Kit – Rat
ChIP-IT® Control qPCR Kit – Human
ChIP-IT® Control qPCR Kit – Mouse
ChIP-IT® Control qPCR Kit – Rat
ChIP-IT® qPCR Analysis Kit
Ready-to-ChIP HeLa Chromatin
Ready-to-ChIP Hep G2 Chromatin
Ready-to-ChIP K-562 Chromatin
Ready-to-ChIP NIH/3T3 Chromatin
Bridging Antibody for Mouse IgG

25 rxns
25 rxns
5 rxns
10 libraries
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
10 rxns
500 µg
1 ml
15 ml
50 rxns

53016
53024
53025
53048
53010
53011
53012
53026
53027
53028
53029
53015
53019
53020
53021
53017
40401
40415
58002

Dounce Homogenizer
ChIP DNA Purification Kit
www.activemotif.com

15

Chromatin Analysis

02

NEXT GEN DNA LIBRARY KITHigh complexity libraries from low cell inputs
The Next Gen DNA Library Kit is designed to prepare Next Generation Sequencing (NGS) libraries from ultra low
DNA inputs, for example from ChIP samples, FFPE samples or cfDNA samples. High efficiency library preparation, combined with molecular identifiers (MIDs), maximizes the amount of unique data obtained from limiting
samples.

✓✓✓✓

Minimize adapter dimer formation, even with just 10 pg input
Maximize unique data using MIDs
Multiplex up to 16 samples for efficient sequencing

TECHNICAL SPECS

Compatible with Illumina sequencing platforms
Fragmented dsDNA

Repair I

Repair steps maximize available DNA.

+ SPRI

Repair II

20 minIn tube wash steps reduce sample loss.

+ PEG/NaCl

Ligation I

15 minIndividual adapter attachment reduces
adapter dimers.

+ PEG/NaCl

Ligation II

10 min+ PEG/NaCl (x2)

Optional PCR

HOW IT WORKS

10 minRecommended for use with:- ChIP DNA- FFPE DNA- cfDNA- limited gDNA samples

Optional PCR Step allows for library amplification, if necessary.

varies+ SPRI

Input:- 100 ng PCR-free- 10 pg with PCR
Procedure length: 2 - 3 hours
Downstream Applications:- ChIP-Seq- WGS- Targeted sequencing

Library

F IGU RE 1: High quality ChIP-Seq
libraries, even with lower cell numbers.
Active Motif’s Low Cell ChIP-Seq protocol was used to prepare chromatinfrom GM12878 cells using the number ofcell equivalents listed. ChIP-Seq libraries
were prepared using either the Next
Gen DNA Library Kit or a competitor kit.
Results show strong peaks that match
ENCODE data sets for the Next Gen
Libraries, even if starting from low cellnumbers.

ORDERING INFORMATION
Product

FormatCat. No.

Next Gen DNA Library Kit
Next Gen Indexing Kit

16 rxns
64 rxns

53216
53264

16

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02

Chromatin Analysis
ChIP NORMALIZATION

Correct for unwanted variation to reveal true biological changes
ChIP is a multi-step process in which variations caused by sample loss during immunoprecipitation and library
preparation, uneven sequencing read depth, or technical variation can lead to results that are difficult tointerpret. To overcome this challenge, Active Motif has developed a ChIP Normalization spike-in strategy fornormalization of ChIP qPCR and ChIP-Seq data to reduce the effects of technical variation and sample
processing bias. This strategy can be applied across samples and antibodies to eliminate bias and reveal true
biological variance induced by causal agents such as inhibitory compounds, mutations, or disease.

✓
✓
✓
✓
✓

Reduce technical variation and sample processing bias
Detect latent or subtle biology not observed with standard ChIP analysis
Can be applied across different antibodies and samples without bias
Works with both qPCR and ChIP-Seq analysis
Can be applied to any ChIP protocol

APPLICATION NOTESChIP normalization can readily be implemented simply by integrating
our spike-in reagents into your standard ChIP protocol as follows:•

A standard ChIP reaction is set up using experimental chromatin(e.g. human) and an antibody of interest.

•

In addition, Drosophila melanogaster Spike-in Chromatin is added, or“spiked-in”, to each reaction as a minor fraction of total chromatin.

•

Spike-in Antibody that recognizes the Drosophila-specific histone
variant, H2Av, is added to the reaction as a mechanism to reliably
pull down the Drosophila chromatin fraction across samples.

ChIP-Seq Normalization Workflow
Experimental
Chromatin

Spike-in
Antibody

Antibody ofinterest

Chromatin Immunoprecipitation

Sequencing

Since variation introduced during the ChIP procedure will also occur
with the Spike-in Chromatin, a normalization factor can be createdbased on the Drosophila signal and applied to the sample genome.
For more complete details, visit us at www.activemotif.com/spikein.

Spike-in
Chromatin

Map to experimental
genomeNormalize sample
tag counts by same ratio

Map to Drosophila
genomeNormalize Drosophila
tag counts across samples

ORDERING INFORMATION
Product

FormatCat. No.

Spike-in Antibody
Spike-in Chromatin
Drosophila Positive Control Primer Set Pbgs
Drosophila Negative Control Primer Set 1

50 µg
15 rxns
96 rxns
96 rxns

61686
53083
71037
71028

www.activemotif.com

17

02

Chromatin Analysis

CHROMATIN ASSEMBLY
Active Motif’s Chromatin Assembly kits & reagents provide a complete and simple solution
for chromatin reconstitution to enable the generation of substrates that closely mimic native
chromatin for use in downstream assays. For more, visit www.activemotif.com/chromassembly.

NUCLEOSOMEASSEMBLY

CHROMATIN ASSEMBLY
KIT

Our Nucleosome Preparation
Kit is designed to prepare monoand oligonucleosomes from cells.
Use to study histones and histone
modifications, enzyme kinetics,
or for inhibitor screening.

Enables in vitro assembly of an extendedarray of ordered nucleosomes from supercoiled
and linear DNA. The resulting chromatin closely
resembles native in vivo chromatin to enablestudies of histone modifications and associatedproteins regulating the target DNA sequence.
Assembled chromatin is ready to use indownstream assays such as in vitrotranscription, ChIP and histone
acetyltransferase (HAT) assays.

NUCLEOSOMEASSEMBLY CONTROLDNARECOMBINANT
NUCLEOSOMES
Pre-assembled humannucleosomes comprised ofoctamers of core histone
proteins (H2A, H2B, H3 & H4)bound by DNA for use in drug
discovery screening (pg 44).

187 bp ds control DNA contains
the well-characterized 601
sequence. Use as a control tovalidate the proper assembly of
your in vitro chromatin assembly
reactions or as a template tomonitor assembly kinetics.

ORDERING INFORMATION
Product

FormatCat. No.

Chromatin Assembly Kit
Nucleosome Preparation Kit
Nucleosome Assembly Control DNA

10 rxns
20 rxns
50 µg

53500
53504
53502

18

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02

Chromatin Analysis
NOME-SEQ

Simultaneously analyze nucleosome occupancy & DNA methylation profiles
Active Motif’s NOMe-Seq (Nucleosome Occupancy and Methylome Sequencing) Kit is the first commerciallyavailable assay to enable the high-resolution analysis of both nucleosome positioning and CpG methylationon the same DNA strand. Currently used methods can only determine either nucleosome occupancy or DNA
methylation state at a specific locus. In contrast, NOMe-Seq is capable of providing genome-wide information
about both epigenetic states simultaneously. Key features & advantages include:✓ Single-molecule resolution analysis of nucleosome occupancy & CpG methylation✓ Determine spatiotemporal correlation between nucleosome positioning and DNA methylation✓ Highly sensitive assay detects even subtle changes in nucleosome position✓ Lacks nucleosome occupancy bias observed with nuclease digestion techniques✓ Chromatin fixation provides information regarding transcription factor binding sites
NOMe-Seq Workflow
Fix cells & shear chromatin by sonication to > 1 kb
CpG sites
GpC sitesCpG sites

NOMe-Seq is listed as one of
the Top Ten Innovations 2013by The Scientist.

Treat with GpC Methyltransferase
CpG sites
GpC sites

TECHNICAL SPECSCpG sites

Input: 750,000 cell equivalents
Conversion efficiency: ≥ 99%

Reverse cross-links/DNA purification

DNA recovery: ≥ 80%

Bisulfite conversion
PCR amplification/cloning, followed by sequencing

Format: Spin column
CpG sites
GpC sites≥ 147 bp
Nucleosome

10-80 bp
ProteinUnmethylated≥ 147 bp
Nucleosome

Methylated≥ 147 bp
Nucleosome

Nucleosome

Elution volume: 50 µl
Procedure length:- GpC methyltransferase: 4 hours- Bisulfite conversion: 5 hours

Bound transcription factor

ORDERING INFORMATION
Product

FormatCat. No.

NOMe-Seq

10 rxns

54000

www.activemotif.com

19

Chromatin Analysis

02

CELL FREE DNA PURIFICATION KIT
Isolate cell-free DNA from human plasma or serum.
Active Motif’s Cell-Free DNA (cfDNA) Purification Kit is designed to isolate cell-free DNA from human plasmaand serum samples. Cell-free DNA can serve as a valuable source of information for non-invasive detection
and monitoring of disease states, however, it is often found in limited amounts in plasma and serum samples.
Using Active Motif’s fast, magnetic bead-based protocol, it is easy to isolate and concentrate cell-free DNA
from fresh or frozen samples.

✓ Works with fresh and frozen samples✓ Process input volumes from 100 µl to 10 ml✓ Fast, magnetic bead-based purification is compatible with automation✓ Low elution volumes to concentrate the cell-free DNA✓ Compatible with Streck Cell-Free DNA BCT® Tubes

Ladder175

353

LadderSample Intensity (RU)

5000

FIGURE 1: TapeStation assessment of the size ofisolated cell-free DNA

4000

Cell-free DNA was isolated from 5 ml of healthy humanplasma using the Cell-Free DNA (cfDNA) Purification Kit. Isolated DNA was run on a TapeStation to assess the size of thepurified DNA. Results show the majority of the purified sample
is cell-free DNA at the expected size of 150-200 bp.

3000
2000
1000

Total yield (ng) / ml plasma

1000
1500

700

500

400

300

200

100

50

25

0

Size (bp)

25
20
15

Cell-Free DNA (cfDNA)
Purification Kit

10

Competitor T

50

Replicate 1

Replicate 2

FIGURE 2: Automation compatibility with KingFisher™
Flex Purification System
The Cell-Free DNA (cfDNA) Purification Kit and a competitor
magnetic bead-based cfDNA purification Kit (Competitor T)
were used to isolate DNA from 600 µl of plasma from a normal
human donor in triplicate using the KingFisher Flex Purification
System. Results show the total DNA yield (ng) per ml plasma.
The Cell-Free DNA (cfDNA) Purification Kit recovered more
DNA than the alternative purification method.

Replicate 3

ORDERING INFORMATION
Product

FormatCat. No.

Cell-Free DNA (cfDNA) Purification Kit

100 ml25503

20

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02

Chromatin Analysis
NU.Q™ TOTAL

Determine total levels of circulating cell-free nucleosomes.
Active Motif’s Nu.Q ™ Total Assay Kit is designed for the detection of global levels of cell-free circulating
nucleosomes (cf-nucleosomes) in human serum in a high-throughput format. For added convenience and amore quantitative interpretation of results, the Nu.Q™ Total Assay Kit also include a recombinant nucleosome
protein for use as a reference standard curve.

✓ Sensitivity: Detect circulating nucleosomes in as little as 10 µl of serum✓ Specificity: Nucleosome epitope specific antibody enables detection of only intact
nucleosomes✓ Quantification: Recombinant nucleosomes enable relative quantification of total levels of
circulating nucleosomes✓ Convenience: Colorimetric assay in a simple 96-stripwell format for high and low throughput✓ Fast: Results can be obtained in 5 hours
Active Motif’s Nu.Q™ Total Assay Kit is powered by VolitionRx.

FIGURE 1: Total circulating cell-freenucleosome detection.
The Nu.Q™ Total Assay Kit was used to assay
human serum samples. The provided Positive
Control was assayed from 0.039 - 2.5 µg/ml asa reference standard curve. Data shown are
the results from wells assayed in duplicate witha 20 minute developing time. These results areprovided for demonstration only.

ORDERING INFORMATION
Product

FormatCat. No.

Nu.Q™ Total Assay Kit

1 x 96 rxns

53510

www.activemotif.com

21

03

DNA Methylation

DNA METHYLATION
Methods for studying and quantifying DNA methylation, such as methylated DNA
enrichment, bisulfite conversion, along with Next-generation sequencing allows researchers
to get a better picture of the genome-wide and gene-targeted changes in DNA
methylation and their association with disease.
Active Motif offers a number of products and services to simplify and streamline DNA
methylation analysis. For more information, visit www.activemotif.com/dna-methylation.

ANTIBODIES &PROTEINS
Comprehensive portfolio of
antibodies & proteins for the
analysis of 5-mC and other
DNA methylation variants (5hmC, 5-fC, 5-caC & 3-mC).

BISULFITECONVERSION
Bisulfite conversion assays forthe analysis of DNA methylation
patterns, genome wide orindividual loci at single basepair resolution

QUANTITATIVEASSAYSMETHYLATED &UN-METHYLATEDDNA ENRICHMENT
A wide selection of assays forenrichment of methylated &hypomethylated DNA for both
5-mC 5-hmC.

22

ASIA: +81 (0)3 5225 3638

Simple, high-throughput ELISAbased assays for measuringchanges in global DNA
methylation levels,hydroxymethylation levels
and DNMT activity.

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation
DNA METHYLATION ANTIBODIESDNA methylation antibodies
Active Motif offers a broad selection of antibodies specific to 5-methylcytosine (5-mC),
5-hydroxymethylcytosine (5-hmC) and other DNA methyl variants for characterization of DNA
methylation. These antibodies have been validated for numerous applications and show nocross-reactivity to non-specific methyl variants or non-methylated cytosines.

F IGU RE 1:  Immunofluorescent images of mitotic chromosome
spreads co-stained with Active Motif’s DNA methylation antibodies.

5-caC
5-mC
DAPI

5-fC
5-mC
DAPI

5-fC
pAb

5-caC
pAb

5-Formylcytosine (5-fC, left) or 5-Carboxylcytosine (5-caC, right)
antibodies (red, Catalog Nos. 61223 and 61225, respectively), a
5-methylcytosine (5-mC) antibody (green) and DAPI (blue) at thetwo-cell stage of mouse preimplantation development. The images
reveal that, at the two-cell stage, only one of the two sister chromatids
is enriched for 5-fC and 5-caC, consistent with findings that 5-fC
and 5-caC levels are diminished by half in blastomeres with eachround of DNA replication (Inoue et al.*).

ANTIBODIES AVAILABLE FOR:✓ 3-mC✓ 5-fC

5-mC
pAb

5-mC
pAb

✓ 5-caC✓ 5-hmC✓ 5-mC

Merge

Merge

F IG U R E 2 : Representative confocal images of fertilized oocytes co-stainedwith Active Motif’s DNA methylation antibodies.
5-Formylcytosine (5-fC) and 5-Carboxylcytosine (5-caC) antibodies (red, Catalog
Nos. 61223 and 61225, respectively), and a 5-methylcytosine (5-mC) antibody(green) (Inoue et al.*).

For a complete list of available
antibodies and detailed information
regarding isotypes, reactivity andvalidated applications, please visit
www.activemotif.com/dnamethabs.

* The images above were kindly provided by the laboratory of Yi Zhang,
HHMI Investigator at the University of North Carolina at Chapel Hill. The data
is described in detail in Inoue et al. (2011) Cell Research 21(12): 1670-1676.

www.activemotif.com

23

DNA Methylation

03

Active Motif also has antibodies specific for proteins that regulate DNA methylation, including
DNA methyltransferase proteins (DNMTs), methyl-CpG binding domain (MBD) protein, and
Ten-Eleven Translocation-1 (TET) dioxygenases. For a complete list of available DNA methylation
related antibodies and detailed information, visit www.activemotif.com/dnamethabs.

Proteins to study DNA methylation
Active Motif offers DNA modifying and restriction
enzymes for DNA methylation analysis, including:+TET• Recombinant TET proteins – convert 5-mC DNA
into 5-hmC DNA for studies of hydroxymethylation(Figure 3). For a list of available proteins, go towww.activemotif.com/tet.

-TET• b-Glucosyltransferase – specifically modifies
5-hmC with the addition of a glucose moiety
to distinguish 5-hmC from 5-mC by the use of
glucosyl-sensitive restriction enzymes. Alternatively, can be used in combination with aradiolabeled UDP-glucose donor for directlabeling of hydroxymethylated residues.

DNA

F IGU RE 3: Dot blot of DNA containing 5-mC shows
the conversion of 5-mC to 5-hmC by Tet1.
DNA containing 5-methylcytosine that was incubatedwith 5 µg of recombinant Tet1 enzyme (+TET) or without
Tet1 (-TET) .

• PvuRts1I restriction enzyme – distinguishes
5-hmC by directly cleaving 5-hmC DNA in
its non-glucosylated form, but will not digest
5-mC or unmethylated cytosine residues. Alsocleaves glucosylated 5-hmC DNA, but at alower efficiency.
• DNA methyltransferase enzymes (DNMTs) – foruse in studies of enzyme kinetics or for screening
enzyme activity in response to compound
treatment (Figure 4).

F IGU RE 4: Recombinant DNMT1 protein activity assay.
Recombinant DNMT1 protein activity was measured using
the DNMT Activity / Inhibition Assay (pg. 34) for 1 hour at 37°C.

ORDERING INFORMATION

24

Product

FormatCat. No.

b-Glucosyltransferase enzyme
PvuRts1I restriction enzyme
Recombinant DNMT1 protein
Recombinant DNMT3A / DNMT3L Complex
Recombinant DNMT3B protein

50 µl
50 Units
20 µg
20 µg
20 µg

55012
55011
31404
31415
31413

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EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation
GLOBAL DNA METHYLATION ASSAYS

Active Motif provides sensitive ELISA-based assays to enable a cost-effective, high-throughput
and accurate method for measuring relative changes in global 5-methylcytosine (5-mC) and
5-hydroxymethylcytosine (5-hmC) levels compared to HPLC or other chromatographic methods.
For more complete information, please visit us at www.activemotif.com/globalmethylation.

Global DNA Methylation – LINE-1 Kit
The Global DNA Methylation – LINE-1 Kit uses a unique hybridization approach to quantify global 5-mC levels thatoffers better specificity & reproducibility than methods utilizing non-specific passive adsorption. The assay is
uniquely designed to quantitate methylation in Long Interspersed Nucleotide Element 1 (LINE-1) repeats inhuman genomic DNA, known as a surrogate marker for global DNA methylation. Kit advantages include:✓ Simple ELISA-based assay to quantify global 5-mC levels in only 5 hours✓ Detects 5-mC from as little as 10 ng DNA and as low as 0.5% methylation✓ Unique LINE-1 probe hybridization method offers better specificity
than passive adsorption

TECHNICAL SPECS
Modification: 5-mC
Input: 10 ng – 200 ng DNA
Readout: Colorimetric✓ Human Jurkat genomic DNA standards provide more biologically
relevant standards for quantification of 5-mC

Adsorption method: LINE-1 hybridization
Cross-reactivity: Human✓ Engineered to provide high specificity, consistency & low background

Format: 96 stripwell microplate
Range of detection: 0.5% – 13%
Procedure length: 5 hours

OD 450nm

2
1.5
F IGU RE 1: Global DNA Methylation Assay.

1

HeLa cells treated with 5-azacytidine (Aza) DNA methyltransferase inhibitor
show decreased 5-mC levels, with standard curve estimating total 5-mC levels
at approximatelt 30% in the untreated and close to 0 in treated cells.

0.5

HeLa + Aza

DNA (100 ng/well)

HeLa

STD 100

STD 75

STD 50

STD 30STD 20

STD 10

STD 0

BLANK

0

ORDERING INFORMATION

www.activemotif.com

Product

FormatCat. No.

Global DNA Methylation - LINE-1 Kit

1 x 96 rxns

55017
25

DNA Methylation

03

Global 5-hmC Quantification Kit
Active Motif has developed the Global 5-hmC Quantification Kit to provide researchers with a robust andquantifiable method to analyze global changes in 5-hmC levels to better understand the influence ofhydroxymethylation on development and disease. The Global 5-hmC Quantification Kit enables you to obtainthe same quantitative results using less sample material and in a higher throughput and easier-to-use format thanmass spectrometry.

✓
✓
✓
✓
✓

Higher throughput, simpler & more cost-effective than LC-MS/MS
Highly sensitive – can detect a little as 0.02% 5-hmC
Can detect 5-hmC from as little as 10 ng of input DNA
Includes DNA standards for quantification of global 5-hmC levels
Highly specific – no cross-reactivity with 5-mC or other methyl variants

0.9

Global 5-hmC ELISA
Mass Spectrometry

0.8% 5-hmC

0.7
0.6

F IGU RE 2: The Global 5-hmC ELISA generates
5-hmC quantification data comparable to mass
spectrometry.

0.5
0.4

Percent 5-hmC in mouse and human tissues determined by the Global 5-hmC ELISA Kit shows equal
quantification as mass spectrometry data using
10-fold less of starting material.

0.3
0.2
0.1
0
mouse mouse mouse mouse
cerebellum heart kidney
liver

mouse
lung

human
brain

human humankidney liver

1.4

TECHNICAL SPECS

Mass Spectrometry% 5-hmC

1.2
1.0

Active Motif's Global 5-hmC Kit

Modification: 5-hmCCompetitor Z Global 5-hmC Kit

0.8

Input: 10 ng – 50 ng DNA

0.6

Readout: Colorimetric

0.4

Adsorption method: Direct detection

0.2

Cross-reactivity: Wide Range0mouse cerebellum

mouse liver

mouse lunghuman brain

Format: 96 stripwell microplate

F I G U R E 3 :  Comparison of various 5-hmC quantification methods.
Percent 5-hmC in mouse and human tissues determined with Active Motif’s Global
5-hmC Quantification Kit show similar levels as determined by mass spectrometry while
Competitor Z’s Global 5-hmC Kit quantification differed significantly.

Range of detection: ≥ 0.02%
Procedure length: 5 hours

ORDERING INFORMATION
Product

FormatCat. No.

Global 5-hmC Quantification Kit

1 x 96 rxns

55018

26

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation

5-METHYLCYTOSINE (5-mC) DNA ENRICHMENT
Choose the DNA methylation kit that best suits your specific research needs
Active Motif’s extensive portfolio of
DNA methylation kits enables you toselect from a variety of enrichment
options. This allows you to customize
features to achieve the results you
desire from your DNA methylation
research. For more information onthese assays, visit
www.activemotif.com/dnamt.

Methylation at:
CpG
non-CpG

DNA Enriched:
ssDNA
dsDNA

Magnetic
Beads

CaptureMethod

MeDIP

✓

✓

✓

–

✓

Antibody

MethylCollector™ Ultra

✓

–

–

✓

✓

ProteinhMeDIP

✓

✓

✓

✓

✓

Antibody

Hydroxymethyl Collector™

✓

✓

–

✓

✓

Chemical

HypoMethylCollector™

✓

–

–

✓

✓

Protein

MeDIP
Active Motif’s MeDIP Kit uses a highly specific 5-methylcytosine (5-mC) capture antibody for methylated DNA
immunoprecipitation (MeDIP). The monoclonal antibody is able to distinguish between 5-mC and 5-hmC,
making this approach more selective than conventional bisulfite conversion or enzymatic methods.

✓
✓
✓
✓

Highly specific antibody enriches only 5-mC methylated DNA
Detects methylated cytosines in both CpG and non-CpG context

TECHNICAL SPECS

Magnetic bead protocol for faster, more efficient enrichment

Modification: 5-mC

One-step IP reaction

Methylation: CpG and non-CpG
Input: 100 ng - 1 µg fragmented gDNA
DNA enriched: ssDNA
Capture method: 5-mC antibody
IP method: Magnetic beads
Procedure length: 2 hours hands-ontime with 1 overnight incubation

F I G U R E 1 : Next-generation sequencing data generated using Active Motif’s MeDIP Kit
correlates well with CpG density.
DNA was enriched from adaptor ligated human PBMC DNA using Active Motif’s MeDIP Kit.
Next-generation sequencing was performed using the Illumina platform and sequencetags were mapped to generate a whole-genome DNA methylation profile. The image
shows that the enriched regions (purple peaks) correlate well with CpG density.

ORDERING INFORMATION
Product

FormatCat. No.

MeDIP

10 rxns

55009

www.activemotif.com

27

DNA Methylation

03

MethylCollector™ Ultra
Active Motif’s MethylCollector Ultra Kit improves the enrichment of CpG-methylated DNA
compared to alternative methyl-binding domain protein (MBD) or antibody IP methods byincorporating the Methylated CpG Island Recovery Assay (MIRA). MIRA uses
a combination of methyl-binding proteins (MBD2b and MBD3L1) to increase
TECHNICAL SPECS
the affinity for methylated DNA. This unique protein complex provides
Modification: 5-mC
greater specificity for methylated CpG dinucleotides than alternative MBD
Methylation: CpG
or antibody immunoprecipitation (MeDIP) techniques in less than half thetime (Figures 2 & 3).
Input: 1 ng - 1 µg fragmented gDNA

✓
✓
✓
✓

Better enrichment than other MBD capture or antibody-based methods

DNA enriched: dsDNA

Magnetic bead-based protocol is completed in < 3 hours

Capture method: Protein (MIRA)

Low input – requires as few as 170 cells (~1 ng DNA) / reaction for qPCR

IP method: Magnetic beads

Magnetic bead protocol for more rapid, efficient & reliable results

Procedure length: < 3 hours

MethylCollector comes in two versions:
MethylCollector™ Ultra Kit – roptimized for identification of methylated CpG DNA
HypoMethylCollector™ Kit– optimized for identification of hypomethylated CpG DNA

F IGU RE 2: MethylCollector Ultra is more sensitive androbust than other MBD enrichment kits.
Comparison of the Active Motif’s MethylCollector Ultra Kit
and a competitor’s MBD-based enrichment kit on human
PBMC DNA. MethylCollector Ultra (top purple panel) identified more methylated regions and the peaks had greater
intensity than peaks identified from the competitor kit (lower
copper panel).

F IGU RE 3: Next-generation sequencing data generatedusing MethylCollector Ultra detects methylation at CpG shores.
DNA was enriched from human PBMC DNA using Active
Motif’s MethylCollector Ultra Kit. Next-generation sequencing
was performed and tags were mapped to generate awhole-genome DNA methylation profile. The data show
one of many examples of DNA methylation detected at
CpG shores rather than in the CpG island itself and agrees
with recent findings showing that methylated sites occur inthese CpG shore regions that are adjacent to CpG islands.

ORDERING INFORMATION
Product

FormatCat. No.

MethylCollector™ Ultra
HypoMethylCollector™

30 rxns
30 rxns

55005
55004

28

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation

5-HYDROXYMETHYLCYTOSINE (5-hmC) DNA ENRICHMENT

Active Motif’s hMeDIP and Hydroxymethyl Collector™ Assays are specific for the enrichment of
DNA containing 5-hydroxymethylcytosine (5-hmC) residues. While most conventional methods
to analyze DNA methylation, such as bisulfite conversion, do not distinguish between 5-mC and5-hmC residues. Active Motif’s hMeDIP and Hydroxymethyl Collector™ enable researchers toisolate 5-hmC DNA separate from 5-mC DNA.

hMeDIP
The hMeDIP Kit contains a highly specific purified 5-hydroxymethylcytosine(5-hmC) antibody and necessary reagents to perform selective immunoprecipitation of either double-strandedor single-stranded genomic 5-hmC containing DNA fragments (hMeDIP). Enriched DNA is ready for downstream
applications including PCR, microarray, or Next-generation sequencing analysis (Figure 1).

✓
✓
✓
✓

Highly specific antibody capture of 5-hmC DNATECHNICAL SPECS

Works with either dsDNA or ssDNA

Modification: 5-hmC

Low input – requires as little as 100 ng DNA/reaction
Includes unmethylated, 5-mC and 5-hmC DNAs & PCR primer controls

Methylation: CpG and non-CpG
Input: 100 ng - 1 µg fragmented gDNA
DNA enriched: dsDNA and ssDNA
Capture method: 5-hmC antibody
IP method: Magnetic beads
Procedure length: 2 hours hands-ontime with 1 overnight incubation

Elevated signal in gene bodies

F IGU RE 1: hMeDIP-Seq performed onhuman brain DNA.
Data shows that 5-hmC is enriched primarily
in the coding region of genes, rather than the
promoter or regulatory regions.

genes + strand

ORDERING INFORMATION
Product

FormatCat. No.

hMeDIP

10 rxns55010

www.activemotif.com

29

DNA Methylation

03

Hydroxymethyl Collector™
Hydroxymethyl Collector utilizes chemical labeling of 5-hmC residues toensure no cross-reactivity with 5-mC, and the method is not limited by the
specific properties or consensus sequences that constrain traditional
methods, such as glucosyl-sensitive restriction enzyme digestion. The result
is reduced background and increased sensitivity to enable enrichment ofDNA fragments containing as few as two 5-hmC residues. Enriched DNA
can be used in the analysis of individual genes by PCR and qPCR, or withmicroarrays and sequencing for genome-wide analysis (Figure 2).

✓
✓
✓
✓

TECHNICAL SPECS
Modification: 5-hmC
Methylation: CpG and non-CpG
Input: 5 ng - 2.5 μg (or 1 - 10 μg for
HMC-Seq) fragmented gDNA
DNA enriched: dsDNA
Capture method: Chemical labeling

Chemical labeling of 5-hmC DNA with no cross-reactivity to 5-mC

IP method: Magnetic beads

Enables detection of non-CpG methylation (CpA or CpT)

Procedure length: < 5 hours

Uses dsDNA for easier library preparation for NGS
Can enrich DNA fragments containing as few as two 5-hmC residues

Hydroxymethyl Collector comes in two versions:Hydroxymethyl Collector™ Kit – recommended for analysis of individual genes by PCR
Hydroxymethyl Collector™-Seq Kit – optimized for use in combination with NGS for 5-hmC genome-wide
analysisF I G U R E 2 : Hydroxymethyl Collector data of enrichment of 5-hmC enrichment across the
JARID2 gene.
Human tiling array data showing enrichment of 5-hmC across the entire length of the JARID2
gene in chromosome 6 using human brain DNA that was enriched with Hydroxymethyl Collector.

ORDERING INFORMATION
Product
Hydroxymethyl Collector
Hydroxymethyl Collector™–Seq Kit
UDP-Azide-Glucose™

30

ASIA: +81 (0)3 5225 3638

FormatCat. No.

25 rxns
25 rxns
1 vial

55013
55019
55020

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation
BISULFITE CONVERSION

Bisulfite Conversion Kit
Active Motif’s Bisulfite Conversion Kit enables  bisulfiteconversion of DNA in as little as 1.5 hours, while minimizing
DNA fragmentation, degradation and sample loss. The included spin columns combine desulfonation and DNA
purification into a single step to quickly generate purified, converted DNA suitable for PCR amplification andsequencing, endonuclease digestion or other downstream applications.

✓
✓
✓
✓
✓

Rapid conversion of DNA in as little as 1.5 hours

TECHNICAL SPECS

Minimal DNA fragmentation, degradation & sample loss

Input: 0.2 ng – 2 µg purified DNA

Thermocycling strategy enables more effective conversion reactions

Conversion efficiency: ≥ 99%≥ 99% conversion efficiency of unmethylated cytosines
Highly sensitive – requires as little as 0.2 ng (50 cells) of input DNA

DNA recovery: ≥ 80%
Format: Spin column
Elution volume: 30 µl

0

20

40

60

80

100

120

140

160

180

200

220

240

260

280

Procedure length: 1.5 hours

Unmethylated CpG
Methylated CpG
F I G U R E 1 :  Bisulfite sequencing results for the CCND2 locus in PC-9 human lungadenocarcinoma cells.
Red circles - methylated CpGs; white circles - unmethylated CpGs.

FFPE Bisulfite Conversion Kit
Active Motif’s FFPE Bisulfite Conversion Kit is designed for DNA methylation
analysis of formalin- or paraformalin-fixed, paraffin-embedded (FFPE) tissues at single-base-pair resolution. This kit provides optimized reagents fortthe recovery of high quality DNA with minimal degradation and greaterthan 99% conversion efficiency.

✓ Minimal DNA fragmentation, degradation & sample loss
✓ Works with up to 4 x 20 µm FFPE tissue sections or 35 mg

TECHNICAL SPECS
Input: 5 pg - 2 µg purified DNA
Conversion efficiency: ≥ 99%
DNA recovery: ≥ 80%
Format: Spin column
Elution volume: 20 µl

unsectioned core samples per reaction✓ ≥ 99% conversion efficiency of unmethylated cytosines✓ Successful bisulfite conversion using 5 pg - 2 µg input DNA

Procedure length: 5 hours

ORDERING INFORMATION
Product

FormatCat. No.

Bisulfite Conversion Kit
FFPE Bisulfite Conversion Kit

50 rxns
40 rxns

55016
55021

www.activemotif.com

31

DNA Methylation

03

ChIP-Bis-Seq
Active Motif’s ChIP-Bisulfite-Sequencing (ChIP-Bis-Seq) Kit combines the target-specific selection of chromatin
immunoprecipitation (ChIP) with bisulfite conversion and sequencing (Bis-Seq) to provide single nucleotide
resolution of immunoprecipitated DNA. By combining the two techniques, information about both the chromatincontext and the methylation profiles of each DNA strand can be evaluated, enabling better understanding of
the biological context and significance regarding gene regulation, gene expression, mechanisms of chromatin
modification and pathway analysis. Key features & advantages include:

TECHNICAL SPECS✓ Interrogate both chromatin and DNA methylation profiles in the same

Input: 1.5- 4.5 x 10^6 cell equivalents

sampleConversion efficiency: ≥ 99%✓ Evaluate allele-specific differences in DNA (imprinting, X-inactivation)

DNA recovery: ≥ 80%✓ Compatible with histone and transcription factor antibodies

Format: Spin column✓ Optimized ChIP protocol provides reduced background caused by non-Elution volume:- ChIP: 36 µl- Library Preparation: 11 µl- Bisulfite conversion: 24 µl

specific binding and higher quality DNA for analysis✓ Reduce sequencing costs by focusing on specific genomic regions ofinterest✓ Save time and money by using our already validated ChIP-Bis-Seq workflow

Procedure length:- ChIP: 2 days
- Library preparation: 2 hours- Bisulfite conversion: 5.5 hours

F IGU RE 2:  DNA methylation profile using
the ChIP-Bis-Seq kit on H3K27me3 enriched
DNA from PC9 cells
Example sequencing results using the ChIPBis-Seq Kit to analyze the DNA methylation
profile of DNA that was enriched for Histone
H3K27me3 in PC9 cells. The top track showsa 423 bp region of chromosome 7 from
Human Hg19 genome. The next track
reveals the ChIP-seq peak profile for Histone
H3K27me3. The final track shows the DNA
methylation profile of forward and reverse
reads as analyzed by bisulfite conversion of
the ChIP-enriched DNA. The blue bars represent unmethylated CpGs, while the red bars
represent CpGs containing methylation.

ORDERING INFORMATION
Product

FormatCat. No.

ChIP-Bis-Seq

10 rxns

53048

32

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

03

DNA Methylation
DNMT ACTIVITY / INHIBITION ASSAY

Non-radioactive assay to screen for DNA methyltransferase activity
Active Motif’s DNMT Activity / Inhibition Assay is a highly sensitive and rapid microplate-based assay to simplify
the measurement of DNA methyltransferase activity or to screen for DNMT
inhibitors without the need for radioisotopes or expensive equipment.
TECHNICAL SPECS
The assay employs a unique and highly sensitive method that detects
Recommended for assaying:
methyltransferase activity using recombinant methyl-CpG binding domain- Purified enzyme activity(MBD) protein that is capable of binding methylated DNA with a higher- Nuclear extract activity- DNMT inhibitors
affinity than antibodies, increasing the sensitivity of the assay relative toantibody-based methods (Figure 1).
Recommended range per well:- Purified enzyme: 0.5 ng - 100 ng- Nuclear extract: 0.5 μg - 10 μg✓ Non-radioactive – colorimetric assay is easily quantified byspectrophotometry on a microplate reader at 450 nm

Readout: Colorimetric✓ Sensitive – activity can be detected from as little as 0.5 ng ofpurified enzyme or 0.5 µg of nuclear extract (Figure 2)

Detection method: MBD2b protein✓ Fast – assay can be completed in < 3 hours✓ Flexible – 96 stripwell microplate for low or high throughput

Adsorption method: Universal CpGenriched DNA substrate

For more information and complete details, please visit us atwww.activemotif.com/dnmt.

Range of detection: 0.5 - 100 ng/well

Format: 96 stripwell microplate

Procedure length: 3 hours

1.4

1.0

1.2
MCF-7 Untreated

0.8

MCF-7 + 5 mM Procaine

OD 450 nm

OD 450 nm

1
0.8
0.6

0.6
0.4

0.4

0.2
0.2

0
0
0.313

0.625

1.25

2.5

5

Blank

10

0.625

1.25

2.5

5

10

Jurkat Nuclear Extracts (µg/well)

Extract (µg/well)

FIGURE 1: DNMT Activity / Inhibition Assay shows
DNMT inhibition in MCF-7 cells treated with procaine,a DNA methyltransferase inhibitor, vs untreated cells.

F IGU RE 2: The DNMT Activity / Inhibition Assay range of detection.
Range of detection is demonstrated with increasing concentrations of Jurkat nuclear
extract prepared using Active Motif’s Nuclear Extract Kit (Catalog No. 40010).

ORDERING INFORMATION
Product

FormatCat. No.

DNMT Activity / Inhibition Assay

1 x 96 rxns

55006

www.activemotif.com

33

DNA Methylation

03

DNA METHYLATION STANDARDS AND CONTROLS
Methylated DNA standard kits
Active Motif offers DNA standards that can be used as controls in methylation analysis
experiments. These include our:
• Methylated DNA Standard Kit – includes 100% unmethylated, 5-mC & 5-hmC recombinant
DNA standards derived from the APC gene promoter. Each standard is 338 bp andcontains multiple cytosine residues in both CpG and non-CpG contexts. In addition to
the DNA standards, PCR primers specific to the APC promoter are also provided (Figure 1).
• 5-Carboxylcytosine DNA Standard Kit – includes two double-stranded DNA oligonucleotides,
one containing a total of 12 5-caC modifications and the other containing only unmodified
cytosine residues (Figure 2).
In addition, Active Motif also provides Jurkat genomic DNA and Fully Methylated Jurkat DNA
for use as controls in DNA methylation analysis experiments.

1

2

DNA

FI GUR E 1 : Dot blot analysis of Methylated DNA
Standard Kit samples.

5-mC

Fifty nanograms of the DNA standards included in
the Methylated DNA Standard Kit were spotted onto
a positively charged nylon membrane:
DNA (unmethylated DNA), 5-mC (5-methylcytosine
methylated DNA), 5-hmC (5-hydroxymethylcytosine
methylated DNA).
Top panel: Dot blot probed with 5-Methylcytosine
antibody (Catalog No. 39649).
Bottom panel: Dot blot probed with 5-Hydroxymethylcytosine antibody (Catalog No. 39769).
Lane 1: single-stranded DNA
Lane 2: double-stranded DNA

5-hmC

DNA
5-mC
5-hmC

5-caC Modified
DNA Standard
ss
ds

Unmodified
DNA Standard
ss
ds

F IGU RE 2:  Dot blot analysis shows specificity of
Active Motif’s 5-Carboxylcytosine antibody for 5-caC.
DNA from the 5-Carboxylcytosine DNA Standard Kit
were spotted onto a nylon membrane to show
specificity of 5-Carboxylcytosine antibody(Catalog No. 61225) for 5-caC.

ORDERING INFORMATION
Product

FormatCat. No.

Methylated DNA Standard Kit
5-Carboxylcytosine DNA Standard Kit
Fully Methylated Jurkat DNA
Jurkat genomic DNA

3 x 2.5 µg
0.5 µg
10 µg
10 µg

55008
55014
55003
55007

ASIA: +81 (0)3 5225 3638
34
www.activemotif.com

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

04

Histone Analysis

HISTONE ANALYSIS
Active Motif offers a wide range of products to analyze histones and histone modifications,as well as the “writers”, “readers” and “erasers” that regulate histone activity. These tools
have been developed for use in basic research as well as clinical and drug discovery
applications. For more, please visit www.activemotif.com/histone.

HISTONE PTM
MULTIPLEX ASSAYHISTONE
QUANTIFICATIONELISAS
Simple, high-throughput
quantitative assays for detecting
global changes in histones andhistone modifications in responseto stimulus, drug treatment or
disease related alterations incell signaling andgene regulation.

HISTONEPEPTIDE ARRAYS
Specificity is a major problem
with histone modification antibodies.
The MODified™ Histone Peptide Arrayenables researchers to screen
antibodies, enzymes and proteinsfor cross-reactivity and specificity.
In addition, the array can be used
to study substrate recognition byhistone-modifying enzymes
and reader domains.

Active Motif partnered with
Luminex®, the industry leader inmultiplexing, to develop the first
epigenetic assay with multiplexing
capability for high-throughput
analysis of histone
modifications.

ENZYME
ACTIVITY ASSAYSHISTONE
PURIFICATION KITS
Proprietary technology
for isolating high quality purified
core histones to enable studies ofchanges in histones and histone
modifications due to disease,
enzymes or drug treatment usingmass spectrometry or similarsensitive quantitative
analysis methods.

www.activemotif.com

High throughput simpleto-use assays to enable
you to accurately andefficiently measure globalchanges in HAT, HDAC
and HDM activity.

35

Histone Analysis

04

MODIFIED™ HISTONE PEPTIDE ARRAY & PRODUCTS
Screen for binding specificity with the broadest coverage of modifications
Active Motif’s MODified™ Histone Peptide Array* offers the most extensive coverage of histone modifications
available from a commercial array for the most accurate screening of binding specificity and cross-reactivityof antibodies and proteins using a simple Western blot-like detection method. Each array contains 384 different
histone modification combinations in duplicate. Modifications include acetylation, methylation, phosphorylationand citrullination on the N-terminal tails of histones H2A, H2B, H3 and H4. Up to four modifications are represented
per 19mer peptide to study not only individual modifications, but also to examine the effects of neighboring
modifications on site recognition and binding. The array is compatible with either ECL-based or colorimetricdetection systems. For more information, visit www.activemotif.com/modified.

✓ Broadest coverage available – 59 histone modifications displayed in 384 unique combinations✓ Study neighboring effects – combines up to four modifications per peptide✓ Simple Western blot-like detection – works with either ECL-based or colorimetric detection

APPLICATION NOTES
Also available as part of our MODified™ products are the:

30

20

10

0

H3

H3

H3

H3

H3

H3

H3

H3

H3

e2

m

K9

e1

m

K9
s

a

e2

s

e2

m

R8

e3

m

R8

e2

m

K4

e1

m

m

K4

K4

ac

K4

a

e2

e2

m

m

R2

R2

Free Array Analyze Software – free PC-compatible software
designed for use with the MODified Histone Peptide Arrayto analyze spot intensities from the MODified array andgenerate a graphical plot of the binding interactions(Figure 1). Information about spot intensity, averages anderrors can be saved in Excel-compatible files. Up to three
individual modifications can be displayed in superposition to
the experimental data for visualization of neighboring effects.

40

H3

•

MODified™ Array Labeling Kit – contains blocking and wash
buffers, rabbit and mouse HRP-conjugated antibodies
and ECL reagents for chemiluminescent detection of the
MODified Histone Peptide Array. For added convenience,
positive control c-Myc antibody is included for recognitionof the array’s control c-Myc tag.

Specificity Factor•

F IGU RE 1:  The MODified Histone Peptide Array confirms
the specificity of modification-specific histone antibodies.
The MODified Histone Peptide Array and MODified Array
Labeling Kit were used to screen Active Motif’s Histone
H3K9me2 antibody (Catalog No. 39683) for cross-reactivity.

* CelluSpots™ arrays are manufactured under license by INTAVIS Bioanalytical Instruments AG and sold through Active Motif as MODified™ Histone Peptide Array.

ORDERING INFORMATION
Product
MODified™ Histone Peptide Array
MODified™ Array Labeling Kit
36

ASIA: +81 (0)3 5225 3638

FormatCat. No.

1 array
5 arrays
5 rxns

13001
13005
13006

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

04

Histone Analysis

MODified™ Protein Domain Binding Kit – screen protein domains for reactivity withhistone modifications
There are several classes of reader domains Domain
Binding Site
Function
Examples(e.g. bromodomains, chromodomains)
Bromo
Acetylated lysine
Regulates chromatin structure and gene expression TAFII250,
that influence gene regulation andresidues
as part of HATs or chromatin remodeling factors
PCAF, GCN5
chromatin remodeling by interacting
Chromo Methylated lysine
Associated with the assembly of protein
HP1b, MPP8,
with specific histone modifications. The
residues
complexes on chromatin
CHD1
MODified Protein Domain Binding Kit is
Tudor
Methylated lysine
It may be involved in RNA binding, DNA damage JMJD2A,
designed for researchers interested inor arginine residues response and chromatin modification
53BP1, SMN
screening these domains for reactivity
Methylated lysine
Unknown, but MBT often appears as repeats in
L(3)MBTL,
MBT
proteins associated with transcriptional repression
residues
CGI-72
with specific histone modifications.
The assay is engineered to be used in
Examples of common protein domains and their binding specificity
conjunction with Active Motif’s MODified
Histone Peptide Array (opposite page). After blocking the MODified Histone Peptide Array with blocking buffer
and adding your His-tagged reader domain of interest, the kit provides all the buffers and reagents needed toscreen binding specificity of the His-tagged reader domain and for chemiluminescent detection of the arrays(Figure 2A). The array image can then be analyzed using the Array Analyze Software (Figure 2B & opposite page).

A

WHAT’S IN THE BOX?

Average Intensity

B 1.0
Addition of modifications at R26
have little effect on binding at K27me3

0.8
Single K27me3
modification

0.6
0.4

Addition of S28phinhibits binding

0.2

od
m
un

K2

H4

H4

H4

K1

H4

0m
6a
K1
e3
2a
c
K2
c
0m
K1
6a
e3
c
K2
0m
e3
H4
K1
6a
c
H4
K1
2a
H3
c
un
m
H3
od
H3
R2
K2
6m
7m
e2
e3
sK
27
m
H3
e3
R2
6m
H3
H3
e2
R2
H3
s
R2
6
6
R2
m
m
e2
6m
e2
a
a
e2
K
27
sK
m
27
e3
m
e3
S2
8p
H3
H3
h
R2
R2
6C
6C
itr
i
H3
t
r
H3
K2
R2
7
K2
6m
7m me
3
e2
e
a
3
S
K2
7m 28p
h
e3
S2
8p
h

0

The MODified Protein Domain Binding Kit contains
interaction buffer, wash buffer, anti-His6-tag
antibody, HRP-conjugated secondary antibody
and ECL reagents for chemiluminescentdetection. For added convenience, His-tagged
recombinant G9a protein, a known histone
methyltransferase that targets Histone H3K9, is
included for use as a positive control.
MODified Histone Peptide Arrays are not includedin the kit and must be purchased separately.

F IG U R E 2 :  The MODified Protein Domain Binding Kit shows the binding
specificity of JMJD2A.
Shown are (A) a representative image of a developed MODified Histone Peptide
Array and (B) data produced by the Array Analyze Software to analyze thebinding specificities of the JMJD2A reader domain to various histone modifications using the MODified Protein Domain Binding Kit and Histone Peptide Array.

ORDERING INFORMATION
Product

FormatMODified™ Protein Domain Binding Kit

5 rxnswww.activemotif.com

Cat. No.
13007
37

Histone Analysis

04

HISTONE PURIFICATION KITS
The only commercial assays available for isolating purified core histones
When using mass spectrometry or similar sensitive quantitative analysis methods to study changes in histones andhistone modifications associated with disease, enzymes or drug treatments, the highest quality samples are required.
Active Motif’s Histone Purification Kits enable you to obtain the purest, highest quality histones from cell or tissue
samples for use in downstream analysis. Active Motif is the only company that offers commercial assays for
purification of core histones. Our proprietary method yields the highest quality samples by improving purity
and preservation of native modifications above crude acid extraction methods (Figure 1).
Active Motif Histone Purification Kits use a unique purification resin and a series of proprietary elution buffers toeliminate contaminating acid-insoluble cellular proteins that are left behind with standard acid extraction
procedures. Removal of these impurities prevents additional enzymatic alterations to histone modifications,ensuring you have the highest quality histone samples for use in your downstream analysis. The results are improved
yield, purity, and preservation of modifications above what is achievable with crude acid extraction (Figure 2).

✓
✓
✓
✓

Unique – only commercial kits available for histone purification
Quality – enhanced histone purity and preservation of histone modifications
Scalability – 3 throughput formats available to suit your needs
Flexibility – works with cell or tissue samples

Acid Extraction Method
SodiumButyrate

Mitotic
ArrestActive Motif Purification Method

Log.

SodiumButyrate

MW

Mitotic
ArrestLog.

Acetylation

T.C.

Brain

MW

98

98

64

64

50

Mitotic
ArrestLog.

SodiumButyrate

Mitotic
ArrestLog.

Methylation

2

50

36

SodiumButyrate

1

36

30

30

H3
16

8.0

4.0

2.0

1.0

0.5

8.0

4.0

2.0

1.0

H4

0.5

Phosphorylation
6

F IG U R E 1 :  Histone purification preserves post-translational modifications
better than standard acid extraction methods.
Active Motif’s Histone Purification method preserves acetyl, methyl andphosphoryl modifications as well or better than crude acid extraction methods.

38

ASIA: +81 (0)3 5225 3638

H2B
H2A

16

H2B
H2B
H2A
H2A

H3
H4

6

F IGU RE 2:  Histone purification isolates fractions of core histones.
SDS-PAGE of histone fractions purified from both tissue and cell samples
using the Histone Purification Kit.

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

04

Histone Analysis

Histone purification kits for quality sample preparation
Achieving the best results is highly reliant on the quality of your input samples. With histone purification, you cansignificantly improve the consistency and reliability of your analysis of histones and their modifications. Purified
histones are ready for quantitative mass spectrometry or other types of analysis, such as in Western blot with
Active Motif’s collection of histone modification antibodies (pages 2-3), for chromatin assembly (page 19)
or as substrates in functional assays, including Active Motif’s Histone Modification ELISAs (Figure 3, page 42).
Total Histone H3 ELISA

3.5

F IGU RE 3: Significantly improve
sample preparation for downstream
applications with histone purification.

3.0
OD 450 nm

2.5

Active Motif’s Histone Purification Mini Kit
and Histone Purification Microplate Kit
produce significantly higher histone yields
than traditional crude acid extraction
methods as measured using the
Total Histone H3 ELISA Kit, page 42.

2.0
1.5
1.0
0.5
0

de

ru
n

t

Ki

io

te

ct

la

tra

Ex

it

p
ro

iK

ic

M

in

M

Recombinant H3 protein (ng/well)C

2.5 1.25 .625 Blank

M

5

A

10

M

20

A

40

Which kit is right for you?
Active Motif’s Histone Purification proprietary histone purification method is available in three different format
options to suit the throughput needs of your downstream analysis. Use Table 1 below to determine which kit isright for you. To learn more, please visit www.activemotif.com/histonepur.
Kit

Histone Purification Kit

Histone Purification
Mini Kit

Histone Purification
Microplate Kit

Application

FormatElutionCapacity# Purifications

Gravity Flow

Separate H2A/H2B &H3/H4 fractions

0.5 - 2.5 mg

10Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.5 - 2.5 mg

10• Mid-range sample
quantities

Mini Spin Column

H2A, H2B, H3 & H4 ina single fraction

0.1 - 0.5 mg

20• High throughput

96 Stripwell Plate
Spin Column

H2A, H2B, H3 & H4 ina single fraction< 0.48 mg

96• Low throughput• Large sample quantities• Medium throughput• Low sample quantities

Specifications for Active Motif’s Histone Purification Kits

ORDERING INFORMATION
Product

FormatCat. No.

Histone Purification Kit
Histone Purification Mini Kit
Histone Purification Microplate Kit

10 rxns
20 rxns
96 rxns

40025
40026
40027

www.activemotif.com

39

Histone Analysis

04

HAT & HDAC ASSAYS
Active Motif’s highly sensitive microplate-based Histone Acetyltransferase (HAT) & Histone
Deacetylase (HDAC) Assay Kits offer a safer and faster alternative to radioisotopic methods
to screen activity or inhibitors of histone acetyltransferases and histone deacetylases fromcell or nuclear extracts, immunoprecipitates and purified enzymes.

Histone Acetyltransferase (HAT) Assay Kit
Arbitrary Fluorescence Units (AFU)The HAT Assay Kit offers a simple fluorescent method to measure
the histone acetyltransferase activity of your samples or toscreen HAT inhibitors. The quick and easy procedure gives you
results in 30 minutes (Figure 1). In addition to optimized buffers
and reagents, the assay includes N-terminal histone H3 andH4 peptide substrates for screening HAT enzymes and apositive control p300 catalytic domain protein to screen forinhibitors. Anacardic acid is also provided for use as a HAT
inhibitor control. A standard curve can be generated with eitherb-mercaptoethanol or Coenzyme A in order to quantitate thefluorescence of your HAT enzyme to determine specific activity.

40K
35K
30K

Histone H3 peptide
Histone H4 peptide

25K
20K
15K
10K
5K
0
Backgroundp300

Anacardic Acid

p300 Activity

F IGU RE 1: The HAT Assay Kit was used to analyze theeffect of anacardic acid, a HAT inhibitor, on p300 activity.

Histone Deacetylase (HDAC) Assay Kit

50K

The HDAC Assay Kit utilizes a peptide substrate that contains
an acetylated lysine residue that can be deacetylated byClass I, IIB and IV HDAC enzymes (Class III HDAC enzymes, or
the Sirtuins, require the addition of the NAD+ cofactor in theassay). Once the substrate is deacetylated, the lysine reacts
with the Developing Solution to produce either a colorimetricor fluorescent product to enable measurement of HDAC activity(Figure 2). A deacetylated assay standard is provided in each
kit to enable calculation of HDAC activity.

Fluorescence Intensity

Trichostatin A
Untreated

40K

30K

20K

10K

0

10

5

2.5

1.25

0.625

0.312

0.156

0

HeLa Nuclear Extract (µg)

F IGU RE 2: Untreated and Trichostatin A-treated HeLa
nuclear extracts were assayed for HDAC activity using thefluorescent version of the HDAC Assay Kit.

ORDERING INFORMATION
Product

FormatCat. No.

HAT Assay Kit (Fluorescent)HDAC Assay Kit (Fluorescent)HDAC Assay Kit (Colorimetric)

1 x 96 rxns
1 x 96 rxns
1 x 96 rxns

56100
56200
56210

40

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

04

Histone Analysis
HISTONE DEMETHYLASE ASSAY

Screen LSD1 for histone demethylase activity
Lysine specific histone demethylase 1 (LSD1, also known as KDM1) specifically demethylates H3K4 and H3K9 andregulates gene silencing during various biological processes, including transcriptional regulation and chromatin
remodeling. Active Motif’s Histone Demethylase Assay provides a simple, high-throughput and accurate method
to screen LSD1 demethylase activity or inhibitor compounds. Assay features include:

✓
✓
✓
✓

Fluorescent assay complete with optimized buffers for measuring LSD1 activity
Use of full-length H3K4me2 instead of peptide substrate more closely resembles in vivo conditions
Includes both positive control LSD1 and a formaldehyde standard for quantitation
Simple, fast microplate-based procedure can be completed in < 2.5 hours

APPLICATION NOTESThe Histone Demethylase Assay is designed to detect formaldehyde released when LSD1 demethylates therecombinant histone H3K4me2 substrate. The formaldehyde by-product then reacts with the Detection
Reagent to generate a fluorescent signal used for quantitation of enzyme activity (Figure 1).
Because the provided recombinant Histone H3K4me2 substrate more closely resembles native histone, theHistone Demethylase Assay enables more accurate measurement of LSD1 demethylase activity. As shown in
Figure 2, the LSD1 enzyme is able to more efficiently demethylate the provided recombinant histone H3K4me2
protein than a histone H3K4me2 peptide substrate. For more complete details, visit www.activemotif.com/lsd1.

% formaldehyde conversion efficiency

80

LSD1 Demethylase Activity

12K

Relative fluorescence

10K
8K
6K
4K
2K

Comparison of Methylated Histone Substrates

70
60
50

73%
40
30
20
10

14%

0

0
Blank

LSD1 Background

LSD1 + H3K4me2 peptide LSD1 + Recombinant H3K4me2 protein

LSD1 + H3K4me2

F I G U R E 1 : Fluorescent detection of LSD1 demethylase
activity using the Histone Demethylase Assay.

F IGU RE 2:  Comparison of demethylase efficiency using theHistone Demethylase Assay shows LSD1 demethylates H3K4me2
protein substrate more efficiently than H3K4me2 peptide.

ORDERING INFORMATION
Product

FormatCat. No.

Histone Demethylase Assay (Fluorescent)

48 rxns

53200

www.activemotif.com

41

Histone Analysis

04

HISTONE MODIFICATION ELISA KITS
Histone H3 trimethyl Lys27 (OD 450nm)

Assay cellular response by quantifying
changes in histone modification levels

1.6

Histone H3 Trimethyl Lys27 ELISA

1.4
1.2

Active Motif’s Histone Modification ELISA Kits provide a
1.0
sensitive, high-throughput method for quantifying global
0.8
0.6changes in the level of specific histone modifications,
0.4
including lysine methylation and acetylation, serine
0.2
phosphorylation, as well as total H3 from purified core
0
0
3.125
6.25
12.5
25
50
100
125
250
5,000 10,000
histones (page 38-39), or histones isolated by acid extraction.
Recombinant Histone H3 trimethyl Lys27 protein
Purified CoreHeLa Acid(provided positive control)
Histones
Extract
Using our expertise in histone antibody development, we
Sample (ng/well)identified optimal antibody pairs for the highly specific and
F IGU RE: The Histone H3 trimethyl Lys27 ELISA was used to assay
sensitive detection of histone modifications and provided
H3K27me3 levels in purified HeLa core histones and HeLa acid extract.
them in a simple sandwich ELISA format for your convenience
Control Recombinant Histone H3K27me3 protein provided in the kit
and ease of use. Each kit includes modification-specific
was assayed as a reference standard curve.
recombinant protein controls to generate standard curves
for quantitation. In addition, the Total Histone H3 ELISA can be used to normalize the amount of histone
modification in your samples when run in parallel with the methylated or acetylated histone ELISAs.

✓
✓
✓
✓

Convenient – colorimetric assays in a simple sandwich ELISA format
Fast – can be completed in < 3 hours
Scalable – stripwell microplate format for manual or high throughput analysis
Quantitative – includes recombinant modified histones for use as standards for quantitation

For an up-to-date list of available Histone Modification ELISAs, please visit www.activemotif.com/hiselisa.

ORDERING INFORMATION
Product

FormatCat. No.

Histone H3 monomethyl Lys4 (H3K4me1) ELISA
Histone H3 dimethyl Lys4 (H3K4me2) ELISA
Histone H3 trimethyl Lys4 (H3K4me3) ELISA
Histone H3 acetyl Lys9 (H3K9ac) ELISA
Histone H3 dimethyl Lys9 (H3K9me2) ELISA
Histone H3 trimethyl Lys9 (H3K9me3) ELISA
Histone H3 phospho Ser10 (H3S10ph) ELISA
Histone H3 acetyl Lys14 (H3K14ac) ELISA
Histone H3 monomethyl Lys27 (H3K27me1) ELISA
Histone H3 trimethyl Lys27 (H3K27me3) ELISA
Histone H3 acetyl Lys27 (H3K27ac) ELISA
Histone H3 phospho Ser28 (H3S28ph) ELISA
Total Histone H3 ELISA

96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns
96 rxns

53101
53112
53113
53114
53108
53109
53111
53115
53104
53106
53116
53100
53110

42

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

04

Histone Analysis

HISTONE H3 PTM MULTIPLEX ASSAY
Simultaneously screen specific and off-target effects on histone modifications
Active Motif partnered with Luminex®, the industry leader in multiplexing, to develop the Histone H3 PTM Multiplex
Assay, the first multiplex epigenetic assay for high throughput profiling of histone post-translational modifications(PTMs) for use with MAGPIX®, Luminex® 200™ or FLEXMAP 3D® instruments. This unique assay enables you to rapidly
and efficiently screen clinical and compound-treated samples and gather more information with smaller input,
in less time and at a lower cost than traditional Western blot, immunofluorescence or chromatographic methods.

APPLICATION NOTES
The microplate-based assay works as a solution-based sandwich
ELISA. Histone modifications in the N-terminus are captured using
antibodies conjugated to fluorescent-labeled magnetic beads.
Each bead emits a unique fluorescent signal to enable detection ofmultiple targets within the same well. A biotinylated H3 C-terminal
antibody is used to capture histones and streptavidin-phycoerythrin(SA-PE) is then added to bind biotin and produce a signal. Fluorescentand SA-PE signals are read using a Luminex instrument and used todecipher both the bead identity and the number of binding events
pertaining to each histone modification. For info on Custom Services,
please go to page 59.

Streptavidinphycoerythrin
Biotinylated
Histone H3 Ab
H3Histonecontaining PTM

SA-PE

tin

tin

Bio

Bio

H3

H3

Ac

Me3

P

PTM Abconjugated bead

12K

What do I need for the assay?

separately to enable customization of analyte selection.
For an up-to-date list of available PTM targets, visit
www.activemotif.com/luminex.

10K
Intensity (MFI)✓ Ab-conjugated Beads for the individual PTMs are soldMedian Fluorescentcontains buffers and reagents for performing the assay.

SPECIFIC EFFECTS

H3K9ac✓ The Histone H3 PTM Multiplex Kit (Catalog No. 33115)

H3 pan-acetyl
8K

6K

-2

-1

0
log[SAHA, µM]✓ Inclusion of Histone H3 Total Ab-conjugated Beads

1

2

H3S10phIntensity (MFI)

8K
Median FluorescentF I G U R E : The Histone H3 PTM Multiplex Assay shows increased histone
acetylation in response to SAHA-mediated HDAC inhibition. Results also show
off-target effects. The dashed lines represent IC50 values for pan-acetyl and H3K9ac.

OFF-TARGET EFFECTS

10K(Catalog No. 33116) in your multiplex enables you tonormalize values for comparison of relative amountsof PTMs across samples.

6K
H3K4me34K

2K

H3K27me3-2

ORDERING INFORMATION
Product

FormatCat. No.

Histone H3 PTM Multiplex Kit

96 rxns

33115

www.activemotif.com

SA-PE

SA-PE

tin

Bio

-1

0
log[SAHA, µM]

1

2

43

HistoneD
rug Discovery
Analysis05

REAGENTS FOR DRUG DISCOVERYOne of the current challenges of epigenetic drug discovery is designing robust enzymatic
assays for high-throughput inhibitor screens. The key to a robust enzymatic assay is obtaining
epigenetic recombinant proteins that are affordable, highly active, and most closely mimic
native enzymatic conditions. Active Motif is a leading provider of reagents, assays, andservices for epigenetic drug discovery. We offer a large collection of full-length high quality
HMTs, HDMs, HATs, HDACs, reader domains and enzyme complexes for many relevant drug
targets, including DNMT1, EZH2, KDM5A, KDM5B, BRD4, PRC2 and MLLs. In addition, we also
provide a wide selection of histone-related substrates (including full-length unmodified
and modified histones and nucleosomes), antibodies and small molecules to provide acomplete solution for designing assays for epigenetic drug discovery.

EPIGENETIC PROTEINS & ANTIBODIES
There is no need to waste valuable time and resources generating
proteins and antibodies for assay development. Active Motif offers acomprehensive portfolio of over 500 ready-to-use purified recombinant
epigenetic proteins, reader domains (visit www.activemotif.com/
proteins, custom and bulk orders available) and over 800 antibodies fordeveloping more efficient epigenetic assays.

WHICH SUBSTRATEIS RIGHT FOR YOU?

HISTONE SUBSTRATES
Results are vastly improved when your assay mimics actual
cellular biology. Active Motif offers a large collection ofrecombinant unmodified & modified histones, histone octamers
and pre-assembled nucleosomes to provide you the best
choice of substrate for use in your assay. For complete details
visit us at www.activemotif.com/recombhis.
Choosing the correct histone substrate for your assay
is key to achieving the best results. Opinion leaders in
epigenetics recognize the power of reconstituting
recombinant chromatin for creating
biologically relevant substrates.

SMALL MOLECULES
Active Motif also offers an expanding collection of small molecule
inhibitors and activators that modulate the activity epigenetic targets,
including methyltransferases, demethylases, acetyltransferases,
deacetylases and bromodomains. For a complete list of available
products, go to our website at www.activemotif.com/smallmol.

44

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

05 Histone AnalysisDrug Discovery
THE ACTIVE MOTIF ANTIBODY PORTFOLIOREADERS✓ Manufacturing in-house, and quality-tested for a range of

WRITERSapplications

bromodomain
chromodomain
PHD finger
WD40 repeat

acetylases
methylases
kinases✓ Available and stocked in 1 mg sizes, ready to ship!
✓ BULK quantities up to 100 mg available upon request+✓ Recombinant mononucleosomes, polynucleosomes, andhistones - containing all relevent modifications

Featured Protiens

ERASERS

Active Motif offers a variety of robust, sensitive, high-throughput
assays for quick and easy screening and profiling of cellularchanges in the activity of histone modifying enzymes. These
include our HAT Assay Kit to measure the activity of histone
acetylases (page 40), the HDAC Assay Kits to analyze histone
deacetylase activity (page 40, Figure 1), and our Histone
Demethylase (HDM) Assay to analyze the lysine demethylase
activity of LSD1 (page 41).

Absorbance (OD405)

deacetylases
demethylases
phosphatases

1.0

Trichostatin A
Untreated

0.8
0.6
0.4
0.2
0.0
50

25

12.5

6.25

3.12

1.56

0.78

0

HeLa Nuclear Extract (µg)

www.activemotif.com

HTRF Signal

HTRF Signal

Active Motif recombinant proteins are manufactured to meet
F IGU RE 1: Untreated and Trichostatin A-treated HeLa
nuclear extracts were assayed for HDAC activity using thethe highest standards of purity and activity required for convencolorimetric version of the HDAC Assay Kit.
tional drug discovery assays, such as HTRF (Figure 2), mass
spectrometry, AlphaLISA and Alphascreen.
These assays are routinely incorporated in
BRD4 (333-460) Titration
BRD4 (44-168) Titration
15K
15K
our quality control process to demonstrate
the nM range activities needed for use in
10K
10K
identification of potent inhibitors. We have
optimized our protein production and testing
5K
5K
to facilitate seamless integration of ourrecombinant proteins into drug discovery
00pipelines. We instill confidence in our proteins
0
50
100
150
200
250
300
0
50
100
150
200
250
300
Bromodomain
conc.
(nM)
Bromodomain conc. (nM)by providing comprehensive application
F IGU RE 2: BRD4 (44-168) and BRD4 (333-460) bromodomains (right) tested by HTRF.
data that is relevant to drug discovery
3.3 μM histone peptide H4K5/8/12/16(tetra-acetyl) was incubated with the protein indicatedplatforms and unparalleled customer
in HTRF assay reaction buffer. Anti-FLAG antibody was used to detect the reaction products.
support for our drug discovery partners.

45Non-coding RNA

06

LIGHTSWITCH ™ LUCIFERASE ASSAY SYSTEM
The LightSwitch™ Luciferase Reporter Assay System provides a comprehensive genome-wide
approach for studying gene element function and identifying gene regulatory networks.
In addition to over 30,000 ready-to-use promoter & 3´UTR reporter constructs, Active Motifalso offers services for custom cloning and mutagenesis, pathway screening, sequencevariant analysis and miRNA target validation. For more detailed information on LightSwitch
products & services, please visit us at www.activemotif.com/lightswitch.

APPLICATIONS:TRANSCRIPTIONAL REGULATION

THE LIGHTSWITCHPRODUCT PORTFOLIO:™

• Over 30,000 pre-cloned humanpromoter, lncRNA promoter & 3´UTR reporters• miRNA mimics and inhibitors• Validated promoter & 3´UTR controls• LightSwitch™ Luciferase Assay reagents• Synthetic miRNA target reporter constructs• Synthetic promoter response elements• Custom Cloning & Mutagenesis Services• Study the mechanisms by which the expressionof a lncRNA gene is induced or repressed• Determine the effect of sequence variations on
lncRNA promoter function• Verify computational predictions and supplement
microarray or Next-generation sequencing data

APPLICATIONS: miRNA /POST-TRANSCRIPTIONAL REGULATION• Validate the 3´UTR targets of any miRNA• Measure the effect of miRNA or siRNA on transcript
stability or translation efficiency• Quantify the impact of a 3´UTR in post-transcriptional
gene regulation• Assess miRNA function in response to various stimuli

46

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

06

Non-coding RNA

LIGHTSWITCH™ LUCIFERASE ASSAY SYSTEM
Genome in - Function Out
With a genome-wide library of over 30,000 cloned regulatory elements, custom cloning andmutagenesis services for those regulatory elements not in the collection, a highly optimized
Renilla luciferase (RenSP) vector system and assay reagents, the LightSwitch™ Luciferase
Assay System is the optimal solution for your gene regulation studies.

LIGHTSWITCHTM REPORTERASSAY SYSTEM
The LightSwitchTM Reporter
Assay System is a complete
solution for performing gene
regulation studies and/or
high-throughput screening.
All LightSwitch vectors
utilize our RenSP luciferase
engineered for gene regulation
experiments to produce higher
levels of luminescence witha shorter half-life than other
Renilla luciferases. LightSwitchTM
reporter vectors provide
industry leading sensitivity anddynamic range which not
only enables you to assess theactivity of regulatory elements,
but also map functional motifs
or characterize the effects ofsequence variation on gene
expression.

Human or other mammalian genomes
EnhancerCloned into
Reporter
Vector//PromoterCloned into
Reporter
RenSP
VectorLuciferase

LightSwitch™
LR Enhancer
Reporter

Gene of interest
RenSP
Luciferase

LightSwitch™
Promoter
Reporter

3´UTR

Cloned into
Reporter
RenSP
Luciferase Vector

LightSwitch™
3´UTR
ReporterChoose a genomic target and vector of interest from ourcollection of over 30,000 ready-to-transfect constructs

Treat withcompounds,
pathogens,or altered
growthconditions

Co-transfect reporter
construct with miRNA
mimics or inhibitors.

miRNA Mimic

RISC
complexTransfect
vectorHybrid mRNA

Light signal

Measure luciferase activity to determine the functional effects of TF binding, treatments, sequencevariations and/or translation of miRNA binding to the hybrid luciferase-3´UTR mRNA

www.activemotif.com

47

06

Non-coding RNA
miRNA TARGET IP KIT
Identify specific mRNA targets of miRNAs

To better understand miRNA-UTR interactions, Active Motif’s miRNA Target IP Kit was designed to capture andidentify the physical interactions of miRNAs with endogenous mRNA transcripts for validation of the binding
targets of specific miRNAs. The targeting of a microRNA (miRNA) to a specific mRNA is mediated through theformation of an RNA Induced Silencing Complex (RISC), containing a combination of various RNA-binding
proteins along with the Argonaute (Ago) protein and miRNA (Diagram 1). The Active Motif miRNA Target IP
Kit utilizes a pan-Ago antibody that recognizes Ago1, Ago2 and Ago3 proteins for immunoprecipitation (IP) of
miRNA/mRNA interactions as part of the RISC complex (Figure 1).

14

miR-122
Non-targeting

Fold Enrichment

12

Mock tfxn

F IGU RE 1: miR-122 targets G6PC3 and SLC1A5.

10

The miRNA Target IP Kit was used on HT1080 cells
transfected with either a miR-122 mimic, a nontargeting miRNA control, or a mock plasmid control.
Following IP using the Ago1/2/3 antibody or Negative
Control IgG included in the kit, qRT-PCR was
performed using primers for G6PC3 and SLC1A5,
which are known targets of miR-122, and GAPDH,
a common housekeeping gene that is not known
to be targeted by miR-122.

8
6
4
2
0
G6PC3

SLC1A5GAPDH

Ribosome
3´UTR
ORF

5´
mRNA

AAA-3´
Ago

miRNA

RISC Complex

DIAGRAM 1: The miRNA within a RISC complex
enables precise silencing of specific mRNA transcripts.
The key components in a RISC complex are an Agoprotein and a miRNA. The Ago protein binds the miRNA,positioning it in a conformation that enables the RISC tobase-pair in a Watson-Crick manner with a mRNA transcript.
This leads to either inhibition of translation (shown) or
increased degradation of the targeted transcript.

ORDERING INFORMATION
Product

FormatCat. No.

miRNA Target IP Kit

10 rxns

25500

48

ASIA: +81 (0)3 5225 3638

EUROPE: +32 (0)2 653 0001

NORTH AMERICA: 877 222 9543

06

Non-coding RNA
RNA SUBCELLULAR ISOLATION KITRNA Subcelular Isolation Kit
Active Motif’s RNA Subcellular Isolation Kit is designed to efficiently isolate separate nuclear and cytoplasmic RNA
fractions for downstream analysis. This method can be used to isolate long and short RNAs, including long noncoding RNAs (lncRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs), PIWI-interacting RNAs (piRNAs), andsmall nucleolar RNAs (snoRNAs) from cells or tissue without cross-contamination or the use of phenolic compounds.

ADVANTAGES
700K✓ Works with cells or tissue✓ Method avoids the use of phenolic compounds✓ Isolates RNA between 15 - 7K nucleotides in size

NuclearReads per million

600K✓ Isolates lncRNAs, miRNAs, siRNAs, piRNAs, snoRNAs,
and other short and long RNAs

✓ Enhanced detection of low abundance transcripts

Cytoplasmic

500K
400K
300K
200K
100K

✓ Reduced background from other intronic or mature RNAs

0
Y-RNA

mRNA

miRNA

piRNA

rRNA

snoRNA✓ Purified RNA is validated for use in RT-qPCR and RNA-Seq
F IGU RE1: The RNA Subcellular Isolation Kit shows small
RNA distribution frequency between cytoplasmic and nuclear
fractions. Cytoplasmic and nuclear RNA were isolated
from HeLa cells using the RNA Subcellular Isolation Kit. RNA
was processed and size selected to remove large RNA. 75
bp single read sequencing was performed and the data
was mapped to miRBase. A frequency of the small RNA(sRNA) distribution for each fraction shows the specificity ofsubcellular isolation.

NuclearRNA

Cytoplasmic
RNA

lncRNA NEAT1
RNA-Seq
Total
RNA

FRM..

NEAT1

RefGeneNuclearRNAF IGU RE 2:  RNA-Seq data confirms lncRNA localization
Nuclear, cytoplasmic and total RNA were isolated from HeLa cells. RNA
was subjected to ribosomal RNA depletion during NGS library preparation. Samples were then sequenced using the Illumina HiSeq and 100
bp paired end reads with 50 M reads per sample. NEAT1 is a lncRNA
that is primarily located in the nucleus, while TUG1 is a lncRNA known tohave both nuclear and cytoplasmic localizations

Cytoplasmic
RNA

lncRNA TUG1RNA-Seq
Total
RNA

MORC2

TUG1

RefGeneORDERING INFORMATION
Product

FormatCat. No.

RNA Subcellular Isolation Kit

30 rxns

25501

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EPIGENETIC & GENE REGULATION SERVICES
The Active Motif Custom Services team makes cutting-edge research accessible to thewider life science community. We provide services for state-of-the-art epigenetics andgene regulation analysis techniques to accelerate your research. For more informationon Active Motif Custom Services, please visit us at www.activemotif.com/services.

ATAC-Seq
ChIP-Seq
RNA-Seq
DNA Methylation Analysis
Histone PTM Quantitation
RIME

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Custom Services
ChIP SERVICES

Choose the global leader in end-to-end ChIP services
Given the importance of ChIP-Seq data sets for development and disease research, obtaining
the highest quality data is crucial. Active Motif offers the most diversified portfolio for ChIP
Services. As the ChIP Experts™, we bring over a decade of experience providing services,with over 15,000 samples processed, and the highest level of expertise of any service provider.

ChIP-Seq Services
Active Motif offers a comprehensive suite ofstandard and specialized ChIP-Seq Services.
Our standard ChIP-Seq Services include:• FactorPath™ ChIP-Seq – map protein-DNA
interactions & histone modifications

Comprehensive
ChIP-Seq Services
1 Customer fixes cell lines
or freezes tissue samples
2 Active Motif prepareschromatin and sonicates
3 Active Motif performs
the ChIP reactions• HistonePath™ ChIP-Seq – genome-wide profilingof histone modifications & interactions• TranscriptionPath™ ChIP-Seq – measure global
transcription rates across the genome• ChIP-qPCR – custom analysis of any gene

Quality Control Steps
to Ensure Success

4 Active Motif constructs
ChIP-Seq libraries
5 Libraries sequenced using
Illumina platform

Chromatin preparation isquantified and sized
Antibody is ChIP Qualified
prior to ChIP-Seq ChIP rxns
qPCR is used to validate
success of the ChIP rxns
Quality control assessment
by quantification of yield
Sequencing to yield≥ 30 million tags

6 Analysis and Data Delivery

In addition, Active Motif offers specialized ChIP-Seq Services that include:• Super-enhancer Profiling – choose from our validated super-enhancer targets• ChIP-Seq Spike-in – a novel ChIP normalization strategy to reveal latent biology• ChIP Antibody Validation – verify if your antibody works for ChIP applications

For more detailed information about Active Motif specialized ChIP-Seq Services,
visit pages 52-57 of the brochure or go to our website at www.activemotif.com/services.

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SUPER-ENHANCER PROFILING SERVICE
Specialized ChIP-Seq service for Super-Enhancer identification & analysis
Active Motif provides comprehensive specialized ChIP-Seq data generation and analysis services for genome-wide
Super-Enhancer profiling. Our end-to-end Super-Enhancer Service* includes sample preparation, ChIP reactions
using ChIP validated antibodies, library construction, sequencing on an Illumina instrument and comprehensive
data analysis. To learn more, visit www.activemotif.com/services-superenhancer.

✓ Identify master regulators of cellular identity✓ Regulatory regions associated with disease✓ Reveal mechanisms of BRD4 inhibitors

3e + 05
2e + 05
1e + 05

H3K27ac Signal at Enhancers

Super-Enhancers

0e + 00

Super-Enhancers are regulatory regions that control
the expression of master transcription factors thatfunction as master regulators of cellular identity.
Misregulation of these transcriptional elements havebeen implicated in diseases such as cancer. Dueto the association with disease, Super-Enhancers
are important drug development targets. Active
Motif utilizes validated antibodies for SuperEnhancer molecular signatures to perform chromatin
immunoprecipitation followed by Next-generation
sequencing to enable the most comprehensive
analysis of these master regulatory domains.

4e + 05

TECHNICAL NOTES

0

5000

10000

15000

Enhancers Ranked by H3K27ac Signal
F IGU RE 1:  Identification of Super-Enhancers.
Enhancers are plotted in increasing order based on ChIP-Seq
peak intensity. Super-Enhancers are the population above theinflection point of the curve.

VALIDATED ANTIBODIES FORSUPER-ENHANCER PROFILING

F IG U R E 2 : BRD4 and H3K27ac ChIP-Seq data identify Super-Enhancers.

✓
✓
✓
✓

✓ H3K27ac
BRD4		✓ OCT4
H3K4me1		
✓ MED1
SOX2		NANOG

The Super-Enhancer is defined by the clustering of high intensity peaks (copper hashes).
This Super-Enhancer is marked by high intensity BRD4 and H3K27ac ChIP-Seq signal.
* Patent No. 9,181,580, Syros Pharmaceuticals.

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Custom Services
ChIP-SEQ SPIKE-IN NORMALIZATION

Novel ChIP-Seq normalization strategy to reveal latent &subtle biological effects
As a leader in ChIP innovation, Active Motif has developed ChIP-Seq Spike-in, an
instrumental technical advancement in ChIP that enables more accurate sample
comparisons and reveals latent biological effects.
Normalization of ChIP data reduces the effects of technical variation and sample
processing bias and is universally applicable to any ChIP experiment. As part of ourcustom ChIP-Seq services, we can apply our ChIP-Seq Spike-in normalization strategy (page 18) across yourChIP samples and antibodies to eliminate bias and reveal real biological differences.

✓
✓
✓
✓

Uncover latent or subtle biology
Monitor consistency between samples
Reduce effects of technical variation
Eliminate sample bias

TECHNICAL NOTESHow does it work?
ChIP-SEQ REACTIONS:•

A standard ChIP-Seq reaction is set up using your experimental chromatin and antibody of interest.

•

Drosophila melanogaster spike-in chromatin is added, or “spiked-in”, to each reaction as a minor fractionof total chromatin.

•

An antibody recognizing the Drosophila-specific histone variant, H2Av, is added to the reaction to reliably
pull down a small fraction of Drosophila chromatin.

•

Following ChIP, immunoprecipitated DNA sequences are analyzed by Next-generation sequencing (NGS).

NORMALIZATION:•

Following NGS, sequence tags are aligned to the reference (e.g. human) and the Drosophila genome.

•

Variances in Drosophila tag counts are equalized across samples.

•

The same ratio used to equalize Drosophila tag counts is applied to human tag counts for normalization.

RESULTS:
Biases introduced during Next-generation library amplification and sequencing also occur in the Drosophila
Spike-in chromatin. Normalization using our Spike-in strategy eliminates these biases to enable the observationof any significant biological changes in your ChIP-Seq samples (reference the ChIP-Seq Normalization Workflow,
opposite page).
For more on ChIP-Seq Spike-in Services or for ordering information, visit www.activemotif.com/services-normalize.

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CHIP-SEQ NORMALIZATION WORKFLOW
A standard ChIP reaction is set up
Experimental
Chromatin

Spike-in
Chromatin

using experimental chromatin andSpike-in
Antibody

Antibody ofinterest

an antibody of interest. Drosophila
Spike-in chromatin and a Spikein antibody that recognizes the

Drosophila chromatin are also
added to the reaction. Since

Chromatin Immunoprecipitation

variation introduced during theChIP procedure will also occur with

Sequencing

the immunoprecipitated Spike-in
Map to experimental
genomeNormalize sample
tag counts by same ratio

chromatin, the Spike-in signal can

Map to Drosophila
genomeserve as a reference to normalize
the test sample signals.

Normalize Drosophila
tag counts across samples

SPIKE-IN NORMALIZATION UNVEILS BIOLOGICAL EFFECTS OF COMPOUND TREATMENTS
Active Motif’s ChIP-Seq Spike-in Normalization strategy reveals EZH2 inhibitor-inducedchanges in H3K27me3 levels that were previously undetected using a standard ChIP-Seq
protocol.*Spike-in Normalization

DMSO

WHY?

_

Without Spike-in normalizaion (-), unevenamplification of the ChIP DNA during
preparation of Next-Gen sequencing libraries
led to loss of differences between samples.
With Spike-in normalization (+) the bias in
PCR amplification was corrected and thedifference between samples is clearly visible.

Inhibitor

DMSO+

Inhibitor*Egan, B. et al. An alternative approach to ChIP-Seq normalization enables
detection of genome-wide changes in histone H3 lysine 27 trimethylation upon
EZH2 inhibition. PLoS One. 11:e0166438

FIGURE: Cells treated with a small molecule inhibitor of EZH2
methyltransferase have dramatic reductions in global H3K27me3 levels.
However, H3K27me3 ChIP-Seq using standard ChIP-Seq protocols (-) does notdetect these differences. Incorporation of Active Motif’s ChIP-Seq Spike-in
Strategy (+) reveals the expected decrease in H3K27me3 ChIP-Seq signal.

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Custom Services
ChIP ANTIBODY VALIDATION

Let the ChIP Experts™ validate the suitability of your antibody for ChIP
One of the greatest challenges in ChIP experiments is the lack of available antibodies that can recognize
fixed, target-bound proteins and that function in immunoprecipitation. Active Motif’s ChIP Antibody Validation
Service makes this process simple, fast, and convenient.

Only 30% of all antibodies work in ChIP-Seq. Therefore, identification of a good ChIP-Seq antibody presents
a significant barrier to project initiation and completion. Our Epigenetic Services team has validated antibodies to over 350 targets. If your target of interest is on our list, we can start your project immediately. Otherwise,
submit an antibody to us and our Antibody Validation Service can give you an answer in as little as 4 weeks.

✓
✓
✓
✓

Submit any antibody for testing‘Yes’ or ‘No’ results for ChIP-Seq antibody performance
Fast turnaround time – results in 4 - 5 weeks.
Hundreds of previously validated antibodies available

ChIP ANTIBODY VALIDATION SERVICES
1

Customer submits fixed
cells or frozen tissue2

Chromatin preparation

3

ChIP reactions

4

ChIP-Seq library
construction

5

Next-Generation
Sequencing

6

Data analysis & delivery

www.activemotif.com

PEAKS Antibody passed its validation.

NO PEAKS Antibody failed its validation.

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GENE EXPRESSION SERVICES
Services for whole transcriptome sequencing & gene expression analysis
Active Motif’s gene expression services include RNA-Seq for identification & quantitation of
RNA transcripts and Pol II ChIP-Seq for measuring transcription rates across the genome.
Combining our Gene Expression Services with ChIP-Seq data enables you to correlatetranscriptional changes with transcription factor & histone modification occupancy.

RNA-SEQ SERVICE

RNA-SEQ SERVICES

Transcriptome profiling by RNA-Seq is a powerful tool that enables the discovery and characterization of novel and existing transcripts and the assessment ofchanges in gene expression in response to treatment or disease. Active Motif
offers comprehensive RNA-Seq Services, from RNA isolation through data analysis,
for any species or sample type. To learn more about our RNA-Seq Service, visit us
at www.activemotif.com/rna-seq.

1 Prepare RNA from
cells or tissues

2 Generate cDNA
3 Construct directional✓ Differential gene experession✓ Changes in gene structure or splicing patterns✓ Effects of TF binding on gene expression

libraries

4 Perform Next-Generation
Sequencing

5 Perform data analysis

POL II ChIP-SEQ SERVICE
Igf1r Expression
6

4
3
2
1
0

✓
✓
✓
✓
✓

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Measure transcription independent of RNA half-life
Identify genes poised for transcriptional activation
Detect alternate start sites and annotated genes
Identify changes in gene expression at early time points
Perform in parallel with targeted ChIP to correlate
occupancy with changes in gene expressionwww.activemotif.com
ASIA: +81 (0)3 5225 3638

Pol II ChIP-Seq
RNA-Seq

5

Fold Induction

Active Motif’s TranscriptionPath™ is a chromatin IP-based assay
to measure transcription rates as a function of genome-wide RNAPolymerase II occupancy. Unlike other RNA-based techniques, themethod measures transcription rates independent of RNA half-life.
The same sample material that is used in your transcription factor
targeted ChIP experiments can be used for the Pol II ChIP-Seq
assay to enable correlation of transcription factor occupancy
with changes in gene expression within the same experiment.
To learn more, visit us at www.activemotif.com/services-pol2.

0

20

40

60

80

100

120

140

160

180 200

MinutesF IGU RE: RNA-Seq and Pol II ChIP-Seq produce
variant gene expression profiles.
Data for Igf1r was extracted from RNA–Seq and RNA Pol II
ChIP-Seq data sets. Cell treatment resulted in induced
gene expression that was measured at various time
points. The cumulative data show that as measured
by Pol II ChIP-Seq, transcription is induced immediately,while mRNA levels only accumulate over time.

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REDUCED REPRESENTATION BISULFITE SEQUENCING
DNA methylation patterns are cell-type specific, and alterations in these patterns can beindicative of disease. Reduced Representation Bisulfite Sequencing (RRBS) is a bisulfite
dependent method that provides single base pair resolution of cytosine methylation at
millions of locations and allowing for sample-to-sample comparisons of DNA methylation
patterns. Comparing DNA methylation profiles from normal and diseased patient samples
can facilitate novel biomarker discovery.
WHY IS RRBS THE RIGHT CHOICE?
RRBS is significantly less expensive than Whole Genome
Bisulfite Sequencing, while still providing the methylation
status of more than 4 million CpGs at biologically relevant
positions such as promoters and CpG islands. You need
only to provide 1 µg of purified DNA, or may also provide
cells or tissues.

FEATURES| Low starting material requirements| Data provided on millions of CpGs
| Data from biologically relevant regions•promoters•CpG Islands

WHY USE RRBS FOR BIOMARKER IDENTIFICATION?
• Single base resolution• Quantitation at each base
• Data at millions of locations across the genome• Data enriched at promoters and CpG islands• Dramatically less expensive than Whole Genome     Bisulfite Sequencing

RRBS SERVICE
Customers submit DNA, cell pellets or frozen tissue then we perform

1 DNA purification
2 DNA digestion
3 Sequencing adaptor ligation
4 Bisulfite conversion
5 PCR amplification
6 Sequencing on Illumina platform
7 Bioinformatic analysis

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FIGURE: RRBS data using biopsied human kidney
tumor and adjacent normal kidney. The displayed
region is a representative region from the genome
wide data set and shows differential DNA methylation
at the promoter of the LAT1 gene. Each block is aseparate data point with red representing a methylated cytosine and blue representing the unmethylated
base.
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DNA METHYLATION ANALYSIS SERVICES
Genome-wide DNA methylation profiling
Active Motif provides comprehensive services for
DNA enrichment and profiling of genomic regions
marked by DNA methylation or DNA methyl variants.
DNA is enriched using our highly validated collectionof DNA methyl or methyl variant specific antibodies
followed by sequencing on an Illumina Next-generation
sequencing platform for genome-wide analysis ofthese modifications.

MeDIP

CpG Density

F IGU RE 1: Genome-wide DNA
methylation analysis.

✓
✓
✓
✓

hmC

Above: Next-generation sequencing
data correlates with CpG density.

Genome-wide services are available for:Right: Genome-wide profiling of DNA
methyl variants.

5-Methylcytosine (5-mC)

fC

5-Hydroxymethylcytosine (5-hmC)
5-Formylcytosine (5-fC)

caC

5-Carboxylcytosine (5-caC)

Targeted Bisulfite Sequencing
NormalPatients72

1

2

DiseasedPatients
3

4

5

6

Disease Model
Cell Line
7

8

9

48

CpG SiteOnce differentially methylated regions are identified
as potential biomarker candidates or regions ofinterest, these regions require further validation
across larger populations. Active Motif’s Targeted
Next-generation Bisulfite Sequencing Service offers asingle base-pair, high-throughput solution for targeted
validation of these regions.

24

Services include:

✓
✓
✓
✓
✓
✓

Bisulfite conversion
Primer design and testing

1

PCR amplification

0

Barcoded library generation
DNA sequencing
Data analysis

50

100

% DNA Methylation
F IGU RE 2: Single base-pair validation of differentiallymethylated sites identified by MethylCollector Ultra-Seq.  
Heat map represents targeted Next-generation bisulfite
sequencing data of 72 CpGs from nine samples.
Differential methylation is observed across the samples.

For a complete list of services, visit www.activemotif.com/servicemethylation.
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Custom Services
MOD SPEC®

Histone modification detection service
Total nuclear levels of histone post-translational modifications (PTM) may differ undervarying conditions – disease vs normal, DMSO vs inhibitor, or WT vs KO. Active Motif’s ModSpec® service can verify expected differences, and more importantly, identify unexpectedchanges in histone PTM levels. This service uses mass spectrometry for relative quantitationof over 80 histone modifications.
QUANTIFY HISTONE MODIFICATIONS USING MASS SPEC
• Optimized to detect over 80 different histone states• Measure acetylation, methylation, ubiquitination, and unmodified peptides• Analyze histone modifications on H1, H2, H3.1, H3.3, and H4• More quantitative and comprehensive than Western blots or ELISA• No hassle. Send your cells to Active Motif and receive data

HOW DOES MOD SPEC® WORK?
HistoneExtraction

Propionylation

Trypsin Digestion

Mass Spec

Analysis

Customers submit DNA, cell pellets or frozen tissue then we perform

Cell pellets or tissues are sent to Active Motif and processed.
1. Histones are acid extracted
2. Lysines are blocked to prevent trypsin cleavage at all lysine aminoacids
3. Histones are digested using trypsin
4. Peptide masses are measured using mass spectrometry
5. Data is analyzed to determine modifications on each histone peptide

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HISTONE PTM QUANTITATION SERVICE
High-throughput multiplex screening of histone modification levels
Active Motif’s Histone PTM Quantitation Service gives youaccess to ground-breaking Luminex® xMAP® magnetic
bead-based multiplexing technology (page 43) to enable
simultaneous measurement of multiple histone modifications
in a single reaction. This first-of-its-kind multiplex epigenetic
assay generates more data using less sample material than
traditional Western blot, mass spectrometry or ELISA methods
and enables you to analyze both specific and off-target
effects in your sample simultaneously.

SERVICE FEATURES✓ MULTIPLEX – analyze multiple histone 		       modifications in a single assay✓ EFFICIENT – use less input amounts than WB or
ELISAs✓ SENSITIVE – 250K cells is sufficient to quantify 13
histone modifications✓ HIGH CONTENT – simultaneously measurespecific & off-target effects

HISTONE PTM MULTIPLEX ASSAY WORKFLOW
Histone-specific modifications on the N-terminus
are captured using antibodies conjugated tofluorescent-labeled magnetic beads. Each beademits a unique fluorescent signal to enable detectionof multiple targets within the same well. A biotinylated
H3 C-terminal antibody is used to capture histones.
Streptavidin-phycoerythrin is then added to bindbiotin and produce a signal. A Luminex instrument
is used to read signals and decipher both thebead identity and the number of binding events
pertaining to each histone modification.

Streptavidinphycoerythrin

SA-PE

Biotinylated
Histone H3 Ab

B

H3Histonecontaining PTM

in
iot

tin

o

Bi

B

H3

H3

Ac

SA-PE

SA-PE

in

iot

P

Me3

PTM Abconjugated bead

F IGU RE: Histone PTM
quantitation data showing EZH2
inhibitor GSK-126 reduction of
H3K27me3 but not H3K9me3
levels.
Acid extracts of HeLa cells
treated for 10 days with 0 - 10
μM GSK-126 were used either
in a three-bead multiplex
assay containing H3-Total,
H3K9me3 and H3K27me3 beads
demonstrate reduction in
H3K27me3 levels.

AVAILABLE TARGETS
H3K4me3H3 pan-ac

H3K9ac

H3S10ph

H3K9me1

H3T11ph

H3K9me2

H3K27ac

H3K9me3

H3K27me2

H3K9me3

H3K27me3

H3K36me3

For more detailed information on our Histone PTM Quantitation Service, visit www.activemotif.com/services-luminex.
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Custom Services
RIME

Mass spectrometry identifies co-factor recruitment into transcriptional  
complexes
Active Motif’s RIME (Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins) Service sheds lighton the complex process of gene regulation by enabling capture and identification of chromatin associatedproteins that interact with an endogenous protein of interest.
WHY RIME?   Gene regulation is often oversimplified when the focus is on one particular transcription factor in any
given cell model. In reality, differential gene expression is greatly influenced by co-factors and other protein interactions within chromatin. RIME clarifies this complexity by providing a means to identify the protein interactions that areimportant for gene regulation.
EXPERIMENTAL DESIGNThe RIME advantage

1.

Antibody validation is performed on asingle sample to show that the target
protein is detected2.

IP-mass spec using the target antibody
is performed in duplicate

3.

IP-mass spec using anti-lgG is
performed in duplicate

4.

IgG interactions are removed from thetarget antibody specific interaction list

✓ Targeting only DNA and chromatin associatedproteins✓ Enabling capture of low affinity interactions✓ Allows more stringent wash conditions resulting inless non-specific interactions

RIME identifies interacting TFs and cofactors

Targeted TF
DNA binding direct interactor
Non-DNA binding direct interactor
Indirect interactor

Ligand 1

Ligand 2

Estrogen Receptor

Estrogen Receptor

Nuclear receptor co-activator 3

Vang-like protein 1

Nuclear receptor interacting protein 1

Pericentriolar material 1 protein

Pericentriolar material 1 protein

Centrosomal protein of 131 kDa

Centrosomal protein of 131 kDa

Protein GREB1

CREB-binding protein

E3 ubiquitin-protein ligase TRIM33

E3 ubiquitin-protein ligase TRIM33

Nuclear receptor interacting protein 1

EXAMPLE DATA FROM RIME: Different Estrogen Receptor(ER) binding profiles have been observed depending on the ligand
used to stimulate ER binding. Our RIME data shows differential
recruitment of co-factors to DNA bound estrogen receptor after
ligand 1 and ligand 2 treatment. Grey indicates recruited proteins
with similar rank order for both ligands. Red indicates common
proteins detected, but with change in order. Purple indicatesunique interacting proteins.

For more information about RIME Services, visit www.activemotif.com/rime.

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BIOINFORMATIC SOLUTIONS
Comprehensive and customizable data analysis & support fromour expert team of scientists
To help our clients interpret large and highly complex whole-genome
data sets, our Services team provides complete bioinformatic analysis
and support for all services. We leverage our extensive experience andbackground to extract relevant information embedded within complex
data sets and provide you with the most meaningful and highest quality
data analysis.

Peptide mapping
Quantitation
Heat map
Clustering
PCA analysis

Mod Spec®
RNA-SeqRaw data
Database searchProtein list
Background removal
Venn diagram
Pathway and GO analysis
Summary report

RIME

Raw data
Sequence quality
Alignment
Peak calling
Data visualization
Differential peak calls
Clustering
Motif discovery
Graphics
Summary report

ChIP-Seq

RRBS
Raw data
Sequence quality
Coverage depth graphic
Data visualization
Methylation at each CpG
Methylation averages
Differential analysis
Heatmap
Clustering
Summary report

Raw data
Data quality check
Alignment
Differential analysis w/ MA plots
Correlation plot
PCA analysis
Heat Maps
Gene Set Enrichment analysis
Summary report

ATAC-Seq

Raw data
Sequence quality
Alignment
Peak calling
Data visualization
Differential peak calls
Clustering
Motif discovery
Graphics
Summary report

For more information, please visit us at www.activemotif.com/services.
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Custom Services
LIGHTSWITCH™ CUSTOM SERVICES

Custom Cloning and Mutagenesis Service
assess the activity of regulatory elements using the LightSwitch system engineredto provide industry-leading sensitivity and dynamic range
In addition to the 30,000 human regulatory elements available as pre-cloned
LightSwitch reporter vectors, Active Motif offers custom services to clone and/or mutate any human, mouse or rat genomic element into any LightSwitch
reporter vector. Services include cloning and sequence validation of every
reporter construct. LightSwitch vectors are engineered to provide optimal kinetics from your reporter assays. For more on LightSwitch™ Custom Services, visit
www.activemotif.com/ls-services.
SERVICE FEATURES• Clone any genomic element of interest

INSERT ANY ELEMENT...

FROM...

INTO ANY
LIGHTSWITCH VECTOR• Promoter, long-range,
3´UTR, and 5´UTR reporter
vectors available

Promoters

Rat

Promoter

Enhancers

Mouse

Long-range• Ideal for gene regulation
studies or validation of
ChIP binding events

UTRs

Human

3’UTR

Motifs

5’UTR

SNP variants• Create sequence variantsof any genomic sequence• Custom vectors ready in
6-8 weeks

For more on Custom Cloning & Mutagenesis Services or to place an order,
please visit www.activemotif.com/ls-services.

IDENTIFY SEQUENCE MOTIFS NECESSARY FOR FUNCTION

FIGURE 1: LightSwitch 3´UTR
Reporter constructs containing

Mutant seed seq

ACACTCCAGTCTCCGNPDA2

ACACTCCAAGTCTCCAANKRD13C

ACACTCCACAGACC

RIMS1

G6PC3

ACACTCCAACAGACCA

G3BP2

ACACTCCGGTCTCC

ALDOA

ACACTCCAAGGTCTCCA

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miR-122 target sites were subjected tosite-directed mutagenesis. Constructs

miRNA luciferase activity

WT seed seq

log2 ratio of targeting/non-targetingthe 3´UTRs of 6 genes known to be0-0.5

were co-transfected in cells withan miR-122 miRNA mimic or non--1

targeting control and then assayed
to identify seed sequences necessary-1.5
Wild-type-2

Mutant-2.5-3

for miR-122 function. Wild-type miRNAs
reduce luciferase expression through3´UTR interactions. miRNA mutationsin the seed sequence disrupt miRNARIMS1GNPDA2 ANKRD13C

G6PC3

G3BP2

ALDOA

targeting and reduce the functional
effect.

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LIGHTSWITCH™ SYSTEM APPLICATIONSTRANSCRIPTIONAL REGULATION• Assess promoter and enhancer response to transcription factor (TF) modulation• Confirm functionality of binding sites identified in ChIP-Seq experiments• Measure functional effects of mutated motifs• Validate computational predictions and add biological relevance to microarray
or NGS data
POST-TRANSCRIPTIONAL REGULATION• Validate the 3´UTR targets of any miRNA, Figure 1• Measure the effect of miRNA or siRNA on transcript stability ortranslation efficiency
VALIDATED PATHWAYREPORTER CELL LINES• Quantify the impact of a 3´UTR in post-transcriptional gene
regulation• Assess miRNA function in response to various stimuli

Target and Pathway Panel

HIF-1aHypoxia

NFkB

Inflammation• Primary screens using the LightSwitch stable cell lines (agonist or
antagonist mode)

AhR

Dioxin, Toxicology

HSF1

Heat shock• Secondary screens using our collection of over 18,000 promoters,
TF response elements, and validated biomarkers

p53

DNA damage, apoptosis

STATInterferon

HIGH-THROUGHPUT SCREENING (HTS)SREBP

Cholesterol biosynthesis• Investigate MOA, specificity, and off-target effects

GR

Glucocorticoid• Dose response analysis

BMP

Bone Morphogenesis

Control

Basal factors

CustomAny pathway or target

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64

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CustomServices

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Products for
Sonication

Histone Modifications
Guide

Stem Cell
Epigenetics

Recombinant Proteins for
Epigenetics Research

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