Gene Regulation
Enabling Epigenetics ResearchTranscription Factor Analysis
RNA Analysis

Products & Services

Gene Regulation

Products & Services

Gene Regulation overview
The Dynamic Process of Gene Regulation
Gene regulation is a highly dynamic process that involves not only transcription factor (TF) and effector
protein interactions with DNA at promoters and enhancer regulatory regions, but also chromatin
remodeling events driven by epigenetic mechanisms. These mechanisms include histone modifications,
DNA methylation, and non-coding RNAs that influence accessibility of transcriptional machinery tounderlying DNA.
All of these mechanisms work in concert to regulate gene expression. Furthermore, once genes aretranscribed, microRNAs and other mechanisms play critical roles in modulating gene expression at thepost-transcriptional level, adding yet another layer of complexity to the process.
Tools to Study Gene Regulation
Active Motif is the industry leader in providing innovative tools to enable epigenetics and gene regulation
research. Our superior products, services, and support serve our life science, clinical, pharmaceutical,and drug discovery partners. Our selection of products to study gene regulation includes TransAM®
transcription factor binding assays and RNA analysis kits and services.
Whether you are already an expert in gene regulation or are just looking to get started, our comprehensive
portfolio of products offers End-to-End solutions to meet the specific needs of your research.

Epigenetic Modifications
Active Chromatin
AcAc

K4

Transcriptional Regulation
Ac K4

K4

Ac K4
CRP
TF

RNA-P

mRNA Transcript

Silent Chromatin
K27

K27

CRPs
K27

K27

K27

Chromatin
RemodelingProteins

TFs

Transcription
Factors

RNA-P

RNA Polymerase
Complex

K27

Post-transcriptional Regulation
K9

K9

K9

K9

K9

Ribosome

K93'UTRHistone Modifications

DNA Methylation

Ac H3K9Ac

K27

H3K27me3

No 5-mC

K4 H3K4me3

K9

H3K9me35-mC

5'mRNA

ORF

AAA-3'

AgomiRNA

RISC Complex

To learn more, visit us at activemotif.com

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

1

Gene Regulation

Products & Services

Transcription Factor Analysis
TransAM® Transcription Factor Activation Assays
TransAM transcription factor assays are fast, user-friendly, and highly sensitive non-radioactive ELISAbased assays that facilitate the study of transcription factor-DNA binding activity in mammalian tissue andcell extracts. TransAM assays are up to 100-fold more sensitive than gel shift assays and also yield morequantitative results.

Advantagesof TransAMTranscription
Factor Assays• Up to 100-fold more sensitive than gel shifts• Results in fewer than 5 hours• Non-radioactive, colorimetric readout• No cloning or transfection required• 96-stripwell format for scalability

Flow chart of the TransAM process.
Activated transcription factor in cell extract binds to the consensus-binding site on the oligo immobilized in the well.
Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activatedtranscription factor.

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

2

Gene Regulation

Products & Services

Our TransAM Assay Kits have been cited in more than 1,000 publications
Select from the list of available transcription factors below and upgrade your transcription factor
activation assays.

Available Transcription Factor Assays

AP-1

IRF-3

ATF-2

MAPK Familyc-Myc

NFATc1

C/EPB

NFkB

CREB & pCREBNrf2

ER

p53

FKHR (FOXO1)

PPARy

GR

Sp1 & Sb1/Sp3

HIF-1

T-bet

HNF

Active Motif offers many different TransAM assays and formats, along with our Nuclear Extract Kit (cat.
nos. 40010 & 40410) for sample preparation and recombinant transcription factors for standard curve
generation.
For a complete up-to-date list of available TransAM products and detailed information, visit us online atactivemotif.com/transam

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

3Gene Regulation

Products & Services

Nuclear Extract Kit
Active Motif’s Nuclear Extract Kit offers a simple method for the preparation of highly pure nuclear,cytoplasmic, and whole-cell extracts from mammalian cells and tissues.
Use of the Nuclear Extract Kit eliminates the need to optimize reagents and ensures consistent sample
preparation. The nuclear extracts generated with this product are of high yield, concentration, and purity,
providing you with the most optimal purified protein fractions for analysis by various methods including
Active Motif’s TransAM® kits, electrophoretic mobility shift assays (EMSAs), and western blots.
Use the Nuclear Extract Kit to improve assay results

Nu WC NP

Easy and efficient sub-cellular fractionation with theNuclear Extract Kit

a-Tubulin
COX IV

Efficiency of cellular fractionation was determined bycollecting 20 µg of nuclear (Nu), and whole-cell (WC)extracts, along with nuclear pellet (NP) using the Nuclear
Extract Kit and performing Western blot using antibodies
against various specific proteins.

GAPDH
Lamin A/CPan-Cadherin

Key Highlights
Fast and Easy - Streamlined
2-hour protocol that requires only
standard laboratory equipment
Scalable - 100 and 400 reaction kit
formats available
Flexible - Prepare nuclear,cytoplasmic, or whole-cell extracts
starting from cells or tissues
Reliable - Quality-controlled
reagents to ensure reproducibility

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

4Gene Regulation

Products & Services

Universal Magnetic Co-IP Kit
The Universal Magnetic Co-IP Kit enables greater flexibility for analysis by Co-IP by providing reagents forisolation of native protein complexes from either nuclear or whole-cell lysates prepared from cells or tissue.
The co-immunoprecipitation procedure utilizes protein G-coated magnetic beads for more rapid andefficient IP and wash steps. The beads also greatly reduce background and minimize sample loss for betterrecovery and improved results from your Co-IP experiments.

Improve your Co-IP
assays with theUniversal Magnetic
Co-IP Kit

• Flexible - Perform Co-IP of nuclear orcytoplasmic protein complexes• Gentle - Preserves protein interactions &modifications• Fast - Magnetic beads streamline
procedure & reduce background• Complete - Includes reagents for both
extraction & IP procedure

By using magnetic beads, it’s possible to use low
stringency buffers while maintaining reduced
background.

Proprietary Enzymatic Shearing Cocktail releases
undissociated protein complexes from the DNA so thateven histone complexes can be studied.

The Universal Magnetic Co-IP Kit was used to makenuclear extract from MCF-7 cells that had been induced
1 hour with 10 nM Estradiol. IP was performed on 300 µg
samples using 2 µg of SRC-1 pAb, ERα pAb and rabbit IgG(as a negative control). Western blot was then performed
using the ERα pAb on 10 µg Input Extract, SRC-1 IP, ERα IP
and the rabbit IgG IP.

HeLa nuclear extracts were made using the Universal
Magnetic Co-IP Kit or a traditional high-salt extraction
protocol, supplemented with 1 µM trichostatin A, adeacetylase inhibitor. Five and ten µg samples of eachextract were used in Western Blot with a Histone H3 acetylpAb. Protein was detected only in samples made using
the kit’s nuclear extraction procedure, as it was designed
to release histone and other protein complexes from DNA
while preserving modifications.

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

5Gene Regulation

Products & Services

RNA Analysis
RNA Analytical Services
Historically the function of RNA in the cell was thought to be primarily a messenger that translated
information from DNA into protein. We now know that RNA is involved in a diverse array of biologicalprocesses including direct catalysis and a multitude of regulatory mechanisms.
Active Motif offers two main gene expression services
Our comprehensive End-to-End RNA analysis services makes it easy and efficient to incorporate genomewide transcriptomic studies into your research.
We offer two main services to investigate gene expression on a genome-wide scale; RNA-Seq for
identification and quantitation of RNA transcripts, and RNA Pol II ChIP-Seq for quantitation of transcription
rates to enable rapid profiling of changes in gene expression associated with transcription factor (TF) andhistone modification occupancy.
RNA Pol II ChIP-Seq Services
Analysis of RNA Pol II occupancy as a proxymeasurement of transcription rates offers theadvantage of enabling you to:RNA-Seq Services
Simply submit RNA, cells, or tissue samples. OrderRNA-Seq alone or combine with ChIP-Seq data to
uncover contextual information about:••••••••••

Measure transcription without the influence of RNA half-life
Identify genes poised for transcriptional activation
Generate gene expression data from cells used for ChIP-Seq
Measure changes at early time points post-treatment
The impact of miRNA inhibitors or stimuli

Differential gene expression
Changes in gene structure or splicing patterns
Effects of TF binding on gene expression
Effect of sequence variants on 3'UTR or miRNA function
The impact of miRNA inhibitors or stimuli

Key Features
RNA Pol II Changes Are Detected Earlier Than mRNA Changes
6Lorem ipsum

5Fold Induction

RNA Pol II ChIP-Seq
RNA-Seq

4

Directional library preparation
Paired-end sequencing on Illumina®
sequencing platform

32
10

PolyA enrichment

020406080100120 140 160 180 200

Gene expression profiles vary depending on theanalysis method.

QC performed using Bioanalyzer
Data analysis pipelines include
differential analysis and GSEA

Data for IGF1R expression was extracted fromRNA–Seq and RNA Pol II ChIP-Seq data sets. Cell
treatment resulted in induced gene expression thatwas measured at various time points. The data showthat transcription, as measured by RNA Pol II ChIPSeq, is induced immediately, while mRNA levels only
accumulate over time.

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

6

Gene Regulation

Products & Services

RNA Subcellular Isolation Kit
Active Motif’s RNA Subcellular Isolation Kit is designed to efficiently isolate separate nuclear andcytoplasmic RNA fractions for downstream analysis. This method can be used to isolate RNA molecules
larger than 75 nucleotides, including long non-coding RNAs (lncRNAs), ribosomal RNAs, messenger RNAs,and small nucleolar RNAs (snoRNAs) from cells or tissue without cross-contamination or the use ofphenolic compounds.

Advantages of the RNA subcellular isolation kit
Works with cells and tissues
Method avoids the use of phenol
Isolates RNA greater than 75 nt, including IncRNAs, snoRNAs, rRNAs,mRNAs, and more
Enhanced detection of low abundance transcripts
Reduced background from other intronic or mature RNAs
Purified RNA is validated for use in RT-qPCR and RNA-Seq

Use the RNA Subcellular Isolation Kit to easily identify
small RNA distributions.

Focus your RNA-Seq on the relevant sub-cellularfractions

Cytoplasmic and nuclear RNA were isolated from HeLacells. 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 thesmall RNA (sRNA) distribution for each fraction shows thespecificity of subcellular isolation.

Nuclear, cytoplasmic and total RNA were isolated from HeLacells. 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 with50 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.

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

7

Gene Regulation

Products & Services

miRNA Target IP Kit
Active Motif’s miRNA Target IP Kit was designed to capture and identify the physical interactions ofmiRNAs 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 the formation of an RNA
Induced Silencing Complex (RISC), containing a combination of various RNA-binding proteins alongwith the Argonaute (Ago) protein and miRNA. The Active Motif miRNA Target IP Kit utilizes a pan-Ago
antibody that recognizes Ago1, Ago2 and Ago3 proteins for immunoprecipitation (IP) of miRNA/mRNAinteractions as part of the RISC complex.

Ribosome
3'UTR
5'mRNA

ORF

AAA-3'

AgomiRNA

RISC Complex

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
to base-pair in a Watson-Crick manner with a mRNAtranscript. This leads to either inhibition of translation(shown) or increased degradation of the targeted
transcript.

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Identify miRNA target genes.
The miRNA Target IP Kit was used on HT1080 cells
transfected with either a miR-122 mimic, a non- targeting
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 miR122, and GAPDH, a common housekeeping gene that is
not known to be targeted by miR-122.

Japan
japantech@activemotif.com

China
techchina@activemotif.com

8

Gene Regulation

Products & Services

RNA ChIP-IT® Kit
Isolate and study RNA-protein complexes specifically from chromatin
To facilitate the characterization of the role of RNA in genome regulation and organization, Active Motif has
applied the efficient magnetic bead-based protocol used to develop our ChIP-IT Express Kits to develop
the first of its kind kit for RNA ChIP. The RNA ChIP-IT Kit was designed to specifically enable investigation ofRNA-protein interactions in a chromatin context and has been optimized for RNA.

Key Features
Specifically tailored to study chromatin-associated RNA
Designed to remove DNA while maintaining RNA integrity
Step-by-step protocols for fixation of chromatin, sonication andimmunoprecipitation, all optimized for RNA preservation
Separate control kit available with control antibody and primers

Study chromatin-associated RNAs.
The RNA ChIP-IT Kit was used on 10 µg samples of DNase
I-treated HeLa chromatin with 10 µl of Suz12 antibody (Cat.
No. 39357) and 2 µg of normal rabbit IgG. The RNA-IP
was performed overnight at 4°C. Real-time RT-PCR wasperformed using primers for the lincRNA SFPQ locus.

Flow chart of theRNA ChIP-IT method

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Japanjapantech@activemotif.com

China
techchina@activemotif.com

9Gene Regulation

Products & Services

Ordering Information
ProductCat. No.

Nuclear Extract Kit

40010

Universal Magnetic Co-IP Kit

54002

Gelshift Chemiluminescent EMSA kit

37341

Nuclear Complex Co-IP Kit

54001

RNA Subcellular Isolation Kit

25501

miRNA Target IP Kit

25500

RNA ChIP-IT® Kit

53024

mTRAP™ Total

23012

mTRAP™ Midi

23024

mTRAP™ Maxi

23006

p53 antibody (mAb)39553

YY1 antibody (pAb)

61779

AbFlex® MAZ antibody (rAb)

91205

AbFlex® MITF antibody (rAb)91201

HIF-1 alpha antibody (pAb)

39665

N6-Methyladenosine (m6A) antibody (mAb)

61755

AbFlex® N6-Methyladenosine (m6A) antibody (rAb)91261

CTCF antibody (pAb)61311

DNMT1 antibody (mAb)61467

p53 antibody (mAb)

39553

AbFlex® RNA Pol II antibody (rAb)91151

RNA pol II CTD phospho Ser2 antibody (mAb)

61083

Recombinant NFkB1 p50 (1-434) protein

81032

Recombinant NFkB1 p105 protein

81143

Recombinant NFkB3 (RELA / p65) protein

81086

Recombinant p53 (TP53) protein

81091

Recombinant YY1 protein

81119

Recombinant MAX protein

81017

Recombinant c-Myc / MAX complex

81087

Recombinant IKKβ protien

81066

Recombinant ALKBH5 protein

31589

Recombinant FTO protein

31572

Recombinant METTL3/METTL14 complex

31570

Recombinant RNA Pol II - CTD protein

81036

Take advantage of Active Motif
Epigenetics Resources!

North America
tech_service@activemotif.com

Europeeurotech@activemotif.com

Epigenetics 101: Complete Guide toUnderstanding Epigenetics
Take a deep dive into epigenetics inour comprehensive learning center.
Epigenetics Blog
Follow our posts about the latest
trends in epigenetics research.
eBooksStay up to date with the latest eBooks
from our epigenetics experts.
Scientific Posters
Download from a collection of ourscientific posters.
Product Literature
Download educational guides, productbrochures, and references lists.
Publications
Search publications that have used
our products and services.
Webinars
Watch the latest tips and techniquesfrom our epigenetics experts.
VideosBrowse our video collection andbecome an epigenetics expert.
Chromatin Clubs& Epigenetics Events
Keep connected to the epigenetics
research community with chromatinclubs, online seminars, virtual
conferences, and more epigenetics
events.
Check out the chromatin club events
that we have scheduled or contact us
to request support for a chromatin club
you are planning.

Japanjapantech@activemotif.com

China
techchina@activemotif.com

10North America
Toll Free:
Direct:Fax:
E-mail:877 222 9543
760 431 1263
760 431 1351
tech_service@activemotif.com

JapanEurope
UK Free Phone:
FR Free Phone:
DE Free Phone:Direct:Fax:
E-mail:0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

China
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com

Direct: +86 21 20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com