ChIP-IT FFPE II(version A1)

Catalog No. 53047

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
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TABLE OF CONTENTS

Page

Overview  . . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Protocol Overview and Time Table . .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  .  . 3Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage
ChIP-IT FFPE II Kit  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols – Experimental Set Up
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Protocols - ChIP-IT FFPE II
Section A. Protein G Agarose Bead Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10
Section B. Pre-clearing the Chromatin  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section C. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section D. Washing & Elution of IP Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Protocols – ChIP DNA Analysis
Section F. Quantitative PCR (qPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section G. ChIP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Appendix
Section H. qPCR Primer Design and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
Section I. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Section J. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

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Overview
Chromatin Immunoprecipitation (ChIP) is a powerful tool for studying protein/DNA interactions,
including transcription factors, co-regulatory proteins, modified histones, chromatin-modifying
enzymes and polymerases because it enables identification of the localization of proteins boundto specific DNA loci. When used in combination with whole-genome analysis such as ChIP-Seq,
insights are possible into gene regulation, gene expression, mechanisms of chromatin modificationand pathway analysis.
Formalin-fixed paraffin-embedded (FFPE) tissue blocks and histology slides are a valuable resource
for retrospective research on clinical samples. Clinical information, treatments and outcomes areoften available for these sample types and large collections of FFPE material is commercially available. The ability to study FFPE samples provides researchers with an opportunity to link FFPE data
to disease, diagnosis and biomarker discovery. Traditionally, FFPE samples have not been useful inchromatin immunoprecipitation because of the limited size of the samples, and the fact that theformalin fixation process often causes degradation and loss of antigenicity.
Active Motif’s ChIP-IT® FFPE Chromatin Preparation II and ChIP-IT® FFPE II Kits are our second
generation FFPE ChIP kits in which the cumbersome de-paraffinization and dehydration procedure
has been streamlined to enable the preparation of high quality ChIP-enriched DNA using lessreagents and hands on time. This new chromatin preparation protocol is optimal when working
with limited amounts of freshly prepared or high quality FFPE tissue (the first generation FFPE
chromatin preparation protocol should be used for precious or highly degraded FFPE samples). The
ChIP-IT FFPE II Kit has been optimized for use after either chromatin preparation using speciallyformulated reagents and protocol guidelines to increase sensitivity and enable analysis by bothqPCR and Next Generation sequencing from extremely limited starting material.
The ChIP-IT FFPE II Kit contains sufficient reagents for 16 immunoprecipitation reactions. It isnecessary to use one of Active Motif’s ChIP-IT FFPE Chromatin Preparation Kits to extract chromatin from teh FFPE samples prior to starting the ChIP-IT FFPE II Kit. The ChIP-IT® qPCR Analysis
Kit (Catalog No. 53029) can be used following ChIP to examine site-specific ChIP enrichment. For
the preparation of Next generation sequencing (NGS) libraries from the ChIP-enriched DNA werecommend that you use the Next Gen DNA Library Kit (Catalog No. 53216) which is 500-fold moresensitive than other library preparation kits. To learn about available ChIP-IT® Control Kits, control
qPCR primer sets, ChIP-Seq validated antibodies, or the EpiShear™ sonication devices, please visit
our website at www.activemotif.com/chip.

product

formatcatalog no.

ChIP-IT® FFPE Chromatin Preparation Kit

5 rxns

53030

ChIP-IT® FFPE Chromatin Preparation II Kit

5 rxns53031

ChIP-IT® FFPE II

16 rxns

53047

Next Gen DNA Library Kit

16 rxns

53216

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1Flow Chart of Process

Prepare agarose beads forpre-clearing and IP reactions

Pre-clear
the chromatin

Prepare chromatin from FFPE slides or tissue blocks
using the ChIP-IT® FFPE Chromatin Preparation or
ChIP-IT® FFPE Chromatin Preparation II Kits
(Catalog No. 53030/53031)

Reverse cross-links, digest
with Proteinase K, and DNA purify.
DNA is now ready for analysis.

Capture antibody-boundprotein/DNA complexes and wash
using ChIP Filtration Columns.

Elute chromatin fromcolumn and save flow-through.

Add primary
antibody of interest.

Flow Chart of the ChIP-IT FFPE II Assay.
The ChIP-IT FFPE II Kit requires chromatin prepared from FFPE slides or tissue blocks using the ChIP-IT FFPE Chromatin
Preparation or ChIP-IT FFPE Chromatin Preparation II Kits (Catalog No. 53030/53031). Chromatin is pre-cleared followed byan incubation with an antibody directed against the DNA-binding protein of interest. The antibody-bound protein/DNA
complexes are immunoprecipitated through the use of Protein G agarose beads and washed via gravity filtration. Following
immunoprecipitation, cross-links are reversed, the proteins are removed by Proteinase K, and the DNA is recovered andpurified. ChIP enriched DNA can be used for either gene-specific or whole-genome analysis.

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2

Introduction
Formalin-fixed paraffin embedded (FFPE) samples represent an opportunity for researchers to
study clinical outcomes of disease and/or treatment conditions in the search of a better disease
understanding, or as a mechanism to identify biomarkers for screening purposes. FFPE samples
serve as the “gold standard” for pathology sample preservation and large collections of thesetissues are available.
There are many challenges associated with working with FFPE samples. The samples are often
limited in size and require the use of multiple histological slides or tissue sections to extract sufficient quantities of material for downstream analysis. Another challenge is the lack of consistency
in the methodologies used for formalin fixation. Some treatments tend to be harsh, causing
degradation of the sample, loss of antigenicity, or they create “overfixed” chromatin which isdifficult to efficiently shear. Although FFPE samples have been used for high-throughput DNA andRNA analysis1,2, the challenges explained above have prevented FFPE material from being used inchromatin immunoprecipitation (ChIP).
ChIP itself can be technically demanding. ChIP requires high-quality antibodies to recognize thefixed, target-bound proteins of interest, and an efficient means to precipitate the antibody/chromatin complex (usually protein A or G beads). In addition, specialized buffers, inhibitor cocktails
and blocking reagents are required to minimize non-specific enrichment and reduce protein
degradation.
Researchers have started to address the need for a methodology to study the influences of epigenetics on normal and tumor samples beyond the traditional immunohistochemistry (IHC) analysis. Pathology tissue chromatin immunoprecipitation (PAT-ChIP) was the first method to extract
and analyze FFPE chromatin for use in high-throughput analysis, such as ChIP-Seq3,4. Subsequently,
many laboratories have attempted to prepare high quality chromatin from FFPE samples5, however
many are still reporting issues in generating quality chromatin from ChIP-seq analysis.
Active Motif has utilized our expertise with ChIP to develop a suite of kits for performing ChIP on
FFPE samples for use in Next-generation sequencing. The ChIP-IT FFPE Chromatin Preparation Kits
contain specially formulated reagents and protocol guidelines to extract high quality chromatinfrom limited amounts of histological slides or tissue sections. Coupled with our newly developed
and most sensitive ChIP kit, ChIP-IT FFPE II, you have everything you need to generate high quality
ChIP-Seq data from extremely limited starting material while producing minimal backgroundsignal, thereby enabling specific detection of the target protein of interest. All Active Motif FFPEChIP kits contain controls to help validate results at each step of the process.

References
1.
2.
3.
4.
5.

Weng, L. et al. (2010) J Pathol., 222: 41-51.
Gu, H.., et al. (2010) Nat. Methods, 7: 133-136.
Fanelli, M. et al. (2010) PNAS, 107(50): 21535-21540.
Fanelli, M. et al. (2011) Nat. Protocols, 6(12): 1905-1919.
Cejas, P. et al. (2016) Nat. Med., 22(6): 685-91.
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3Kit Performance and Benefits
ChIP-IT FFPE II Advantages:• Sensitive enrichment of DNA from nanogram quantities of FFPE chromatin• Optimized reagents reduce background levels caused by non-specific binding events• Works with both transcription factor and histone ChIP-validated antibodies• Filtration based washes are the easiest wash method available and result in increased consistency in multi-sample experiments• Highly robust procedure has been validated using FFPE chromatin from both normal andtumor samples with proven performance in both qPCR and ChIP-Seq analysis• Includes positive control antibody to help confirm chromatin quality and successful IP

Detection limit: The ChIP-IT FFPE II Kit requires a minimum of 200 ng chromatin extracted andvalidated using the ChIP-IT FFPE Chromatin Preparation or ChIP-IT FFPE Chromatin Preparation II
Kits (Catalog No. 53030/53031) for use in each ChIP reaction. Using larger quantities of chromatin
is recommended when available in order to ensure good enrichment efficiency, but due to thelimited size and yield of FFPE material, it may not be possible to obtain more than 200 ng ofchromatin for use per ChIP reaction.
Product Performance: The ChIP-IT FFPE II Kit relies on the use of chromatin extracted fromFFPE samples. It is important to understand the caveats of working with FFPE chromatin and thepotential pitfalls. Due to the variability that exists in the formalin fixation process and the storage
conditions of the sample, not all FFPE material may yield high quality chromatin. Some fixation
treatments tend to be harsh, causing degradation of the sample or loss of antigenicity. This loss ofantigenicity may result in reduced ChIP efficiency. Additionally, certain FFPE samples may be moredifficult to physically homogenize and sonicate during chromatin preparation. A Histone H3K9ac
antibody (Catalog No. 91104) is included in the ChIP-IT FFPE II Kit as a positive control antibody tohelp confirm success of the ChIP reactions. Successful chromatin immunoprecipitation depends on
the quality of the chromatin preparation, reatined antigenicity, the affinity of the ChIP antibody
and the abundance of the target protein.

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4ChIP-IT® FFPE II

Figure 1: qPCR analysis of Glioblastoma samples assayed using the ChIP-IT FFPE II Kit
Samples of glioblastoma patient-derived xenografts were processed using the ChIP-IT FFPE Chromatin Preparation II Kit.
Three 5 µm curls were used for Sample GBM1, and cell pellets from dissociated tumor were used for Samples GBM2 and
GBM3. Samples were reverse cross-linked, proteinase K treated and purified for analysis by qPCR using the included DNA
standards and qPCR primer sets to determine the quantity and quality of the extracted chromatin. If sufficient quantities
are not obtained after a single sonication, additional rounds of sonication can be performed (as specified in Section F).

Figure 2: H3K27ac and H3K27me3 ChIP-Seq on Glioblastoma samples.
Samples of glioblastoma patient-derived xenografts were used in H3K27ac and H3K27me3 ChIP-Seq to generate genomewide profiles of this histone modification. Libraries were constructed using the Next Gen DNA Library Kit (Catalog No.
53216). A portion of that data is presented above.

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5Kit Components and Storage
The ChIP-IT FFPE II Kit is for research use only. Not for use in diagnostic procedures. All components can be stored at -20°C prior to first use. Then we recommend storing each component at
the temperatures indicated in the tables below.
Reagents

Quantity

Storage

Proteinase K (10 µg/µl)

75 µl

-20°C

10X PBS450 µl-20°C

100 mM PMSF

160 µl-20°C

Protease Inhibitor Cocktail (PIC)

80 µl

-20°C

Carrier

32 µl

-20°C

Blocker

80 µl

-20°C

Blocking Reagent AM1

112 µl-20°C

BSA (10 mg/ml)

112 µl-20°C

Protein G Agarose Beads*

1.12 ml

4°C

Histone H3K9ac rAb

8 μl

-20°C

Human Positive Control Primer Set GAPDH-2 350 μl-20°C

Human Negative Control Primer Set 2

350 μl-20°C

TE pH 8.0

33.6 mlRT

5 M NaCl

350 µlRT

ChIP Filtration Columns

16 ea

RT

ChIP Buffer

45 ml

RT

Wash Buffer AM1

29 ml

RT

LiCl Buffer

29 ml

RT

Elution Buffer AM4

1.6 mlRT

1.7 ml siliconized tubes

32 ea

RT

Low EDTA TE

1 ml

RT

* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their first use.
Once thawed, the Protein G beads should not be re-frozen by the customer. Protein G Agarose
Beads should be stored at 4ºC.

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6

Additional materials required• A ChIP-Seq-validated antibody directed against the protein of interest• Phenol, saturated (DNA Purification, Molecular Biology Grade, Amresco Catalog No. 0945)• Chloroform/isoamyl alcohol (24:1) (DNA Purification, Molecular Biology Grade)• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Apparatus to rotate tubes end-to-end at 4°C (e.g. a Labquake from Barnstead/Thermolyne
with a tube holder for 1.5 ml microcentrifuge tubes)• Microcentrifuge (table top centrifuge 4°C) and microcentrifuge tubes• 250 µl PCR tubes• Thermal cycler• Pipettors and tips (filter tips are recommended)• (Optional) ChIP-IT® qPCR Analysis Kit (Catalog No. 53029)• (Optional) SYBR Green qPCR master mix (Bio-Rad Catalog No. 170-8882)• (Optional) Next Gen DNA Library Kit (Catalog No. 53216)

Protocol Overview and Time Table
Required Time
Protein G Agarose Bead Preparation*3 - 6 hours (Pre-clearing beads) to overnight (IP beads)

Pre-clearing the Chromatin*

2 - 3 hours

Immunoprecipitation

Overnight incubation

Binding to Protein G agarose Beads

3-4 hours

Wash Immune Complexes & Elute DNA

45 minutes

Reversal of Cross-links

Overnight incubation

DNA Purification

1.5 hours

qPCR Analysis

2 hours* Some steps may be performed concurrently.

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7

Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Buffer Preparation
100 mM PMSF and Protease Inhibitor Cocktail (PIC)
Thaw the PMSF and the PIC at room temperature until fully dissolved, which takes about
30 minutes. Vortex gently and spin down briefly before use, then add to the buffers immediately
before use.
ChIP Buffer
Is supplied ready to use.
Protein G Agarose Beads
For best results, gently shake and invert the tube to resuspend the agarose beads. The beads settle
quickly, and therefore should be resuspended just before pipetting. We recommend cutting 2 mmfrom the end of a pipet tip prior to pipetting to prevent the tip from becoming clogged. Protein
G Agarose Beads are shipped on dry ice and can be stored frozen until their first use. Once
thawed, beads should not be re-frozen by the customer. Protein G Agarose Beads should be
stored at 4ºC.
1X PBS Buffer
Prepare a 1X PBS solution by adding 1 ml 10X PBS to 9 ml sterile water. Mix by inverting and place
on ice to chill. 1X PBS Buffer can be prepared in large quantities and stored at 4ºC for 6 months.
Histone H3K9ac rAb
Active Motif’s Histone H3K9ac rAb (Catalog No. 91104) is included as a ChIP-validated positive
control antibody. This antibody can be used to confirm the ChIP reactions are working with your
FFPE chromatin material. Histone H3K9ac is a mark of transcriptional activity and should be highly
abundant in all sample types. Use 2-4 µl of antibody per ChIP reaction.
Human Positive and Negative Control qPCR Primer Sets
Active Motif’s Human Positive Control Primer Set GAPDH-2 (Catalog No. 71006) and Human
Negative Control Primer Set 2 (Catalog No. 71002) are included to confirm the enrichment of
the positive control Histone H3K4me3 pAb from human chromatin samples. To confirm Histone
H3K4me3 enrichment using a different species, please see Active Motif’s list of available Control
qPCR primer sets at www.activemotif.com/chipprimers. The primers are provided at a concentration of 2.5 µM. Use 4 µl of each primer set per qPCR reaction.

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8

Recommendations
ChIP-Seq-validated Antibody
We recommend using 2-4 µg antibody per ChIP reaction in a maximum volume of 30 µl. However,this will vary according to the affinity of the antibody and the quality of the chromatin; you may
need to use more of a particular antibody. ChIP antibodies must recognize fixed, native proteinthat is bound to DNA and/or complexed with other proteins. Many antibodies that perform wellin other applications do not perform in ChIP. Thus, ChIP performed with an antibody that has notbeen ChIP-Seq-validated must include appropriate controls (such as Active Motif’s ChIP-IT Control
qPCR Kits, Catalog Nos. 53026, 53027 and 53028) to validate the chromatin preparation and theChIP methodology. To see a list of available ChIP-validated antibodies available from Active Motif,
please visit www.activemotif.com/chipabs.
Chromatin Quantity
It is recommended to use a minimum of 200 ng chromatin prepared using Active Motif’s ChIP-IT
FFPE or ChIP-IT FFPE II Chromatin Preparation Kit (Catalog No. 53030/53031) for each ChIP reaction
in a total ChIP reaction volume of 200 µl. When possible, using larger quantities of chromatin isrecommended in order to ensure good enrichment efficiency. However, due to the limited size
and yield of FFPE material, for some sample types it may not be feasible to obtain more than
200 ng. We suggest using qPCR quantitation, or a Qubit™ Fluorometric Quantitation Method todetermine the concentration of the FFPE chromatin. These methods are more sensitive at detecting low DNA quantities and will provide more accurate results than a spectrophotometer such as
Nanodrop. If the chromatin concentration is too dilute to utilize 200 ng in a 200 µl ChIP reaction
volume, we suggest preparing multiple ChIP reactions at 200 µl and then pooling the enriched
DNA together during the DNA purification step. It is not recommended to perform ChIP reactions
in larger volumes as the increased volume of the reaction reduces the enrichment efficiency.

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9

Protocols
Section A. Protein G Agarose Bead Preparation1.

Set up two 1.5 ml microcentrifuge tubes to prepare the Protein G Agarose beads for the IP
and pre-clearing. We recommend cutting 2 mm from the end of a pipet tip prior to pipetting
Protein G agarose beads to prevent the tip from becoming clogged. If more than 12 IP reactions will be performed, we suggest setting up multiple tubes to ensure sufficient room inthe tube for the addition of all the required components. Volumes to add will be based on
the number of IP reactions performed. Include negative and positive control IPs into the calculation for the number of IP reactions. Volumes shown below include excess for pipetting.
		 IP Reactions			Pre-clearing Reactions
Reagents
4 rxns 8 rxns 12 rxns
4 rxns 8 rxns 12 rxns
Protein G Agarose

140 µl270 µl400 µl140 µl270 µl400 µl* We do not recommend more than 12 IP reactions in a single tube. If more than 12 IPs are to be
performed in a single experiment, set up multiple tubes.

2.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

3.

Add the calculated volume of TE, pH 8.0 to the each tube of beads:
		 IP Reactions			Pre-clearing Reactions
Reagents
4 rxns 8 rxns 12 rxns
4 rxns 8 rxns 12 rxns
TE, pH 8.0

460 µl900 µl 1330 µl

460 µl900 µl1330 µl

4.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

5.

Set up bead blocking reactions according to the table below:
		 IP Reactions			Pre-clearing Reactions
Reagents
4 rxns 8 rxns 12 rxns
4 rxns 8 rxns 12 rxns
TE, pH 8.0

126 µl243 µl360 µl140 µl270 µl400 µlBlocking Reagent AM1 14 µl

27 µl

40 µl

14 µl

27 µl

40 µl

BSA

14 µl

27 µl

40 µl

14 µl

27 µl

40 µl

Blocker

14 µl

27 µl

40 µl

–

–

–

Incubate at 4°C		
with rotation

Overnight			

3 - 6 hours

6.

Cap tubes and incubate on an end-to-end rotator at 4°C. Follow the recommendations in thechart above for the incubation time.

7.

Following the incubation, proceed to Section B with the pre-clearing bead reactions.
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10Section B. Pre-clearing the Chromatin
1.

If chromatin is stored at -80°C, place on ice to thaw.

2.

Remove the pre-clearing reactions from the rotator and quick spin to collect contents tothe bottom of the tube. Allow the IP beads to continue to incubate on the rotator.

3.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

4.

Add the calculated volume of ChIP Buffer to the pre-clearing reaction. Mix by inverting.
		 IP Reactions			Pre-clearing Reactions
Reagents
4 rxns 8 rxns 12 rxns
4 rxns 8 rxns 12 rxns
ChIP Buffer

–

–

–

460 µl900 µl1330 µl

5.

Centrifuge the tubes at 1250 x g for 3 minutes. Carefully remove and discard supernatant,taking care to avoid the beads.

6.

Perform a second wash with ChIP Buffer using the volumes below. Mix by inverting.
		 IP Reactions			Pre-clearing Reactions
Reagents
4 rxns 8 rxns 12 rxns
4 rxns 8 rxns 12 rxns
ChIP Buffer

–

–

–

160 µl

315 µl465 µl7.

Cut off 2 mm from the end of pipet tip and transfer 50 µl of the pre-cleared Protein G
agarose bead slurry to the 1.7 ml siliconized tube containing the sonicated chromatin from
Section C, Step 3.

8.

Add 5 µl Protease Inhibitor Cocktail and 5 µl PMSF to each tube.

9.

If using less than 200 µl chromatin, adjust the final volume of the reaction to a final volumeof 260 µl using ChIP Buffer.

10. Cap tubes and incubate on an end-to-end rotator for 2-3 hours at 4°C.

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11Section C. Immunoprecipitation1.

Prepare separate, labeled 1.5 ml microcentrifuge tubes for each ChIP reaction, even if thesame antibody will be used for more than one sample. Prepare the antibody mixture according to the table below and place on ice.
Reagent

Antibody Mixture

Antibody

2-4 µg

1X PBSUp to 30 µl final volume2.

Centrifuge the tubes containing the pre-clearing reactions (Section B, Step 10) in a microcentrifuge at 3500 rpm for 3 minutes. Carefully transfer the supernatant, avoiding the beadpellet, to the microcentrifuge tube containing the appropriate antibody mixture.

3.

Cap tubes and incubate on an end-to-end rotator overnight at 4°C.

4.

The next day, remove the IP reactions (from Section A, Step 5) from the rotator and quick
spin to collect contents to the bottom of the tube.

5.

Add 50 µl of the IP Reaction bead slurry to each immunoprecipitation reaction. We recommend cutting off 2 mm from the end of a pipet tip to make it easier to pipet the bead slurry.

6.

Cap tubes and incubate on an end-to-end rotator for 3-4 hours at 4°C.

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12Section D. Washing & Elution of IP reactions
1.

Label a ChIP Filtration column for each ChIP reaction. Remove the tab from the bottom of
the column and place in an empty 1 ml pipet tip box as a holder.

2.

Remove the immunoprecipitation reactions from the rotator and quick spin to collect contents to the bottom of the tube.

3.

Add 600 µl ChIP Buffer to each immunoprecipitation reaction to wash any remaining beads
off the sides of the tube and transfer the entire volume (including the Protein G agarose
beads) to its labeled column. Allow flow-through to occur by gravity.

4.

During the gravity flow, transfer 100 µl Elution Buffer AM4 per ChIP reaction to a new 1.5 ml
microcentrifuge tube. Pre-warm at 37°C. (DO NOT add to columns until Step 10 below.)5.

Wash each column with 900 µl ChIP Buffer. Allow a minute for the buffer to flow throughthe column. Wash a second time with an additional 900 µl ChIP Buffer.

6.

Wash each column with 900 µl Wash Buffer AM1. Incubate for 3 minutes on the column.
Allow the buffer to flow through the column. Wash a second time with an additional 900 µl
Wash Buffer AM1.

7.

Wash each column with 900 µl LiCl Buffer. Incubate for 3 minutes on the column. Allow thebuffer to flow through the column. Wash a second time with an additional 900 µl LiCl Buffer.

8.

Wash each column with 900 µl TE Buffer pH 8.0. Allow a minute for the buffer to flow
through the column. Wash a second time with an additional 900 µl TE Buffer pH 8.0.

9.

Transfer columns to a new 1.5 ml microcentrifuge tube. Centrifuge at 1250 x g for 3 minutes atroom temperature to remove residual wash buffer.

10. Label new 1.5 ml microcentrifuge tubes. Transfer the ChIP Filtration Columns to the labeledtubes. Add 50 µl pre-warmed (37°C) Elution Buffer AM4 to each column. Incubate at room
temperature for 5 minutes. Centrifuge at 1250 x g for 3 minutes at room temperature to collect ChIP DNA.
11.

Keeping the columns in the same microcentrifuge tube, add an additional 50 µl pre-warmed(37°C) Elution Buffer AM4 to each column. Incubate at room temperature for 5 minutes.
Centrifuge at 1250 x g for 3 minutes at room temperature to collect ChIP DNA

12.

Discard the ChIP Filtration columns. The flow through contains the ChIP DNA.
www.activemotif.com

13Section E. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube. Add 2 µl Proteinase K and 5 µl 5 M NaCl.
Vortex to mix. Heat in a thermocycler at 65°C overnight.

2.

Following reversal of cross-links, transfer DNA to a new 1.5 ml microcentrifuge tube. Add
125 µl phenol and 64 µl chloroform/isoamyl alcohol (24:1). Shake vigorously for 15 seconds tomix (do not vortex). Incubate at room temperature for 5 minutes.

3.

Centrifuge in a microcentrifuge at maximum speed for 2 minutes. Transfer the aqueous layer(top layer) to a new 1.5 ml microcentrifuge tube. A hazy white layer at interface may still bepresent.

4.

Add 125 µl chloroform/isoamyl alcohol (24:1) to the aqueous layer in the new tube. Shake vigorously for 15 seconds to mix (do not vortex). Incubate at room temperature for 5 minutes.

5.

During the incubation, set up new 1.5 ml microcentrifuge tubes for each ChIP reaction andadd 2 µl Carrier to each tube.

6.

Centrifuge the DNA purifications in a microcentrifuge at maximum speed (> 18,000 x g) for 2
minutes. Transfer the aqueous layer (top layer) to microcentrifuge tube containing Carrier.

7.

Add 300 µl 100% ethanol to the DNA solution and briefly vortex to mix.

8.

Place samples at -80°C for 30 minutes to precipitate the DNA.

9.

Centrifuge in a microcentrifuge at maximum speed (> 18,000 x g) for 15 minutes at 4°C. Markthe tube where you expect the pellet to form as it may not be visible.

10. Carefully remove the supernatant with a pipet. Avoid disturbing the location of the DNA
pellet.
11.

Add 500 µl 70% ethanol to each tube and invert to mix.

12.

Centrifuge in a microcentrifuge at maximum speed (> 18,000 x g) for 5 minutes at 4°C. Markthe tube where you expect the pellet to form as it may not be visible.

13.

Remove 400 µl supernatant and centrifuge a second time at maximum speed (> 18,000 x g)
for 2 minutes at 4°C. Carefully remove as much residual ethanol as possible with a P200 pipet
taking care not to disturb the pellet.

14.

Air-dry the pellet for 10-15 minutes (until residual ethanol has evaporated). Resuspend thepellet in 40 µl Low-EDTA TE Buffer. ChIP DNA is ready for analysis by qPCR or NGS library
preparation, or may be stored at -20°C for future use.

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14

Protocols – ChIP DNA Analysis
Section F. Quantitative PCR (qPCR)
ChIP DNA can be analyzed on a gene-specific basis using qPCR. Positive control and negative
control PCR primer pairs should be included in every analysis to determine the fold enrichment.
Negative control primers will amplify a region of the genome not bound by the antibody target ofinterest. Active Motif recommends the use of its ChIP-IT® qPCR Analysis Kit (Catalog No. 53029),
which contains positive and negative control primer pairs, standard curve DNA, standard curve
primers and a qPCR Analysis spreadsheet to perform the qPCR analysis calculations. Active Motif’s
analysis strategy determines primer efficiencies and the ChIP sample values are normalized according to input, primer efficiency, chromatin amount used in the ChIP reaction and resuspension
volume. If not using the ChIP-IT qPCR Analysis Kit, qPCR data normalization and graphing can bedone using the methods described in Section H.
1.

Below is an example qPCR reaction. Please follow the specific instructions for your qPCR
instrument. We recommend using a commercially available SYBR Green qPCR master mix(e.g. Bio-Rad Cat # 170-8882) and preparing triplicate reactions. If the ChIP elution volume
was reduced for ChIP-Seq analysis, please see the notes under General Recommendations
on page 16 to dilute the ChIP DNA for use in qPCR. Input DNA that was prepared in the
ChIP-IT FFPE Chromatin Preparation or ChIP-IT FFPE Chromatin Preparation II Kit (Catalog No.
53030/53031) and used to quantify the chromatin extracted from the FFPE samples should
also be included.
Reagent

20 µl PCR reactions

2X SYBR Green master mix

10 µl

Primer mix (2.5 µM each primer)*

4 µl

Sterile water

1 µl

DNA sample (ChIP or Input)

5 µl

Total volume

20 µl* We recommend designing primers to perform at an annealing temperature of 58°C so thatall qPCR reactions can be performed under identical conditions. An amplicon length of
75-150 bp is recommended. Active Motif offers validated species-specific qPCR primers
designed according to these recommendations at www.activemotif.com/chipprimers.
2.

Place tubes in a real time PCR instrument and program as below:

		

95°C for 2 minutes(95°C for 15 seconds, 58°C for 20 seconds, 72°C for 20 seconds) for 40 cycles

3.

Include and inspect the melt curve based on the protocols recommended by the qPCR
instrument manufacturer to ensure that primer pairs amplify only a single product.

4.

If using the ChIP-IT qPCR Analysis Kit, please refer to the product manual for analysis instructions. Otherwise, follow the recommendations in Section H.
www.activemotif.com

15Section G. ChIP-Seq
The ChIP-IT FFPE II Kit has been validated for whole genome analysis using the Next Gen DNA
Library Kit (Catalog No. 53216), designed to prepare Next Generation Sequencing (NGS) libraries
from double-stranded input DNA for use with Illumina® sequencing platforms. Libraries can begenerated from as little as 10 pg high quality genomic DNA, ChIP DNA, FFPE DNA or cell-free DNA(cfDNA), which is 500-fold more sensitive than other library preparation kits. Additionally, libraries
can be prepared from 100 ng DNA if preparing PCR-free libraries. The Next Gen DNA Library Kit
offers the advantage of including molecular identifiers (MIDs) during library generation to enable removal of true PCR duplicates from the sequencing analysis. This helps to distinguish PCR
duplicates from fragmentation duplicates, thereby, increasing the number of unique alignments
for more accurate data analysis.
We strongly recommend that you use the Next Gen DNA Library Kit to prepare your libraries forsequencing due to its high sensitivity. However, if using alternative sequencing platforms, please
select an appropriate library preparation method compatible with the sequencing apparatus to be
used.
A. General Recommendations• Verification of the quality of the ChIP-enriched DNA should be performed prior to proceeding with downstream sequencing. Follow the instructions in Section F: Quantitative PCR(qPCR) with the following modification: the ChIP DNA will need to be diluted prior to use inqPCR due to the reduced elution volume. Dilute 5 µl of ChIP DNA in 45 µl sterile water. Use 5µl of the diluted ChIP DNA per qPCR reaction.
• For good ChIP-Seq data, quality enrichments with low background is more important than
the total quantity of DNA recovered. Therefore, we recommend performing qPCR on known
binding sites to verify enrichment levels against a negative control primer set using Active
Motif’s ChIP-IT qPCR Analysis Kit (Catalog No. 53029) to confirm DNA quality rather thanquantifying the DNA. Quality DNA that is suitable for use in ChIP-Seq should show enrichment of known binding sites over negative control primer sets of 5-fold (see Figure 4 onpage 19). If the qPCR enrichments are satisfactory then the remaining 35 µl of ChIP DNA canbe used for library generation.
• A typical high quality ChIP-Seq reaction will yield 10-20 million unique alignments. The
number of unique alignments is highly correlated with the amount of starting ChIP DNA that
goes into the library preparation reaction. Therefore, as the amount of ChIP DNA decreases,
the number of unique alignments in the ChIP-Seq data set will decrease. Due to the limited
size of FFPE samples, there is usually a low amount of starting material available for ChIP and
therefore, subsequent low amounts of ChIP-enriched DNA. This means the library complexity can be less than in a typical ChIP-Seq data set where >5 million cells may have been used
per ChIP reaction. FFPE yields will be in the range of 2 million to 10 million unique alignments.
Although these results are not as robust as a typical ChIP-Seq data set, these data sets canstill be reliably interpreted. The interpretation will be easier for targets that yield tight peaks
as compared with targets that produce broad peaks. Data sets may be improved by increaswww.activemotif.com

16

ing the amount of FFPE material used in ChIP, by pooling multiple ChIP reactions together
for library generation or by increasing the sequencing depth. Despite the reduction in thenumber of unique alignments, FFPE ChIP-Seq is enabling the generation of data sets thatwere not possible to obtain in the past.
• 36 bp single end reads are sufficient for unique mapping and good ChIP-Seq data, although
longer reads can be used
• Input DNA should be sequenced as a control reaction in order to identify false “peaks” andalso to reveal regions of the genome that have been duplicated. Subtracting the input peaks
from the experimental peaks will help to eliminate false data. Use 50 ng Input DNA for
library generation.

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17

Appendix
Section H: qPCR Primer Design and Data Analysis
A. Design of the primers• Design and analyze your potential primer pairs using an in silico PCR program (i.e. Primer3
at http://frodo.wi.mit.edu/ or the UCSC Genome Browser at http://genome.cse.ucsc.edu/
cgi-bin/hgPcr).
• Primers that dimerize should be avoided, as they will be bound by SYBR Green, which willcompromise accurate quantitation. You can test your primers for self-complementarity andsecondary structure at http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi.
• Ideally, the amplicons should be 75-150 bp in length.
• For use with the ChIP-IT qPCR Analysis Kit, primers should be designed to anneal optimally at
58°C with a recommended length of 18-22 bp.
• Active Motif offers ChIP Control qPCR primer sets validated to work in our ChIP-IT qPCR
Analysis Kit. To see a list of the available species-specific primers, please visit
www.activemotif.com/chipprimers.
B. Data Analysis
If the data analysis will not be performed using Active Motif’s ChIP-IT qPCR Analysis Kit, two othersimplified methods of analysis are provided below. Both methods listed require the generationof a standard curve, containing known amounts of DNA, for each primer pair being used in theexperiment.
Method 1: Fold enrichment of positive primers relative to negative control primers
1. Produce a standard curve by performing qPCR with your primer set on known DNA
quantities of Input DNA in triplicate. Run three to five samples that are 10-fold dilutions, e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.
2.

Run the ChIP and IgG samples along with the dilution series of the Input DNA standards using both positive control primers (known binding sites) and negative control
primers (regions of the genome not bound by your protein of interest).

3.

Your qPCR instrument will assign values to each qPCR reaction based on the standard
curve. If your machine does not average your triplicate reactions automatically, you willneed to calculate these averages.

4.

Divide the average value from the positive control primer set by the average value ofthe negative control primer set to obtain your fold enrichment.

Method 2: Express data as a percent of input
1. Produce a standard curve by performing qPCR with your primer set on known DNA
quantities of Input DNA in triplicate. Run three to five samples that are 10-fold dilutions, e.g. 0.005 ng, 0.05 ng, 0.5 ng, 5 ng and 50 ng.
www.activemotif.com

18

2.

Run the ChIP and IgG samples along with the dilution series of the Input DNA standards using both positive control primers (known binding sites) and negative control
primers (regions of the genome not bound by your protein of interest).

3.

Your qPCR instrument will assign values (in ng) to each qPCR reaction based on thestandard curve. If your machine does not average your triplicate reactions automatically, you will need to calculate these averages.

4.

For each qPCR reaction you will have used a percentage of your total ChIP DNA. In
order to calculate the amount in the whole reaction, divide the elution volume ofthe entire ChIP reaction by the volume used in the qPCR reaction (e.g. if you eluted
ChIP DNA in 100 µl and used 5 µl in the qPCR reaction the formula is 100/5 = 20). Then,
multiply the average qPCR quantity by this number (e.g. qPCR quantity in ng x 20).

5.

To express data as a percent of input, divide the adjusted values from Step 4 above by
the amount of DNA that went into the ChIP reaction and then multiply by 100%. (e.g.
if 20 µg was used in the ChIP reaction this is equivalent to 20,000 ng of chromatin. The
calculation would be the adjusted value from Step 4 divided by 20,000 ng and thenmultiplied by 100). Typical percent of input recovered values are 0.05% to 1%.

www.activemotif.com

19Section I. Troubleshooting Guide
Problem/question

Recommendation

At what points in the
protocol can I stop?

The protocol may be stopped and samples stored at the times and temperatures below:
1. During overnight incubation of pre-cleared chromatin and antibody, 4°C.
2. During overnight reversal of cross-links, 65°C
3. After DNA clean up, -20°C.

Performing ChIP with alarge volume of chromatin.

This is not recommended. It is better to set up several small ChIP reactions (240 μl each)and pool the samples at the end, rather than trying to ChIP a single large sample. Do not
perform a single scaled-up reaction, as the capture efficiency is lower.

High background.

Chromatin not sheared enough. Shearing should produce DNA fragments that are smallenough to exclude background from neighboring chromosomal sequences, but still large
enough that there is a good possibility your amplicon remains intact. We recommend
200-1200 bp fragments. If the DNA fragments are too large, the background is increased.
Consider increasing the number of pulses for sonication.
Antibody issue. Too much antibody relative to the amount of chromatin in the ChIP reaction. Excess antibody will result in more non-specific binding, which will be detected asincreased background. We recommend using 2-4 µg of antibody per IP.

Poor or no enrichmentwith target antibody.

Too little chromatin. Generally, we recommend using greater than 200 ng of chromatin
per ChIP reaction. If your ChIP antibody has low binding affinity and you have limited
chromatin for the ChIP, you may obtain low enrichment. Try and repeat the ChIP reaction
using more chromatin. Be sure to quantitate the concentration of the sheared chromatin
sample(s) being ChIP’d to ensure both that adequate chromatin is used per sample, andthat equal mass quantities of chromatin are used in each ChIP. Due to the low yields ofchromatin from FFPE samples, using a Qubit® Fluorometric Quantitation or qPCR quantification method is recommended over a Nanodrop since the Nanodrop does not have theaccuracy to read very low DNA quantities.
Antibody is not ChIP validated. The antibody does not efficiently recognize fixed
proteins, either because the epitope is destroyed by fixation or because the epitope ismasked by other proteins in a larger complex. To assist in ChIP validating an antibody, it isvery useful to use a positive control antibody such as Histone H3K9ac (Catalog No. 91104)
and a negative IgG from the same species, and primers that have been proven to workin the type of PCR being used. Active Motif offers species-specific ChIP-IT Control qPCR
Kits for antibody validation (Catalog Nos. 53026, 53027 & 53028).
Low-affinity antibody. Use a different antibody.
Antibody affinity to protein G is weak. Individual monoclonals have variable binding
affinities to protein G, which are pH dependent; the optimal pH may vary for each Ig, For
those with low to medium affinity, capture efficiency by protein G can be dramatically
improved through use of our Bridging Antibody (Catalog No. 53017). This antibody is arabbit anti-mouse pAb that recognizes all subclasses of mouse immunoglobulins. If yourIgG has a weak/medium affinity to protein A or G, the Bridging Antibody will increase
antibody capture by the beads without increasing background.
Problems with PCR. Confirm the amplified sequence for the positive control primer set isbound by the antibody target. Identify other binding sites.

No PCR products for the
ChIP’d samples (but the
Input DNA yields thecorrect PCR product)

Increase the amount of chromatin used in the ChIP reaction, the amount of antibody
used, or both.

No PCR products withreal-time PCR

Confirm the species specificity and efficiency of your primers. You may need to redesign
your primers. Primers that work in end point PCR do not always work in qPCR.

www.activemotif.com

Use a different antibody.

20

Section J. Related Products
ChIP-IT® Kits		

FormatCatalog No.

ChIP-IT® Express		ChIP-IT® Express Enzymatic		
ChIP-IT® Express Shearing Kit		
ChIP-IT® Express Enzymatic Shearing Kit		
Low Cell ChIP-Seq Kit		
ChIP-IT High Sensitivity®		ChIP-IT High Sensitivity® Chromatin Preparation Kit		
ChIP-IT® qPCR Analysis Kit		
ChIP-IT® ChIP-Seq		
ChIP-IT® FFPE Chromatin Preparation II Kit		
ChIP-IT® FFPE Chromatin Preparation Kit		
ChIP-IT® Express HT		
Low Cell ChIP-Seq		
Next Gen DNA Library Kit		
Re-ChIP-IT®		RNA ChIP-IT®		
Chromatin IP DNA Purification Kit		
EpiShear™ Probe Sonicator		
EpiShear™ Cooled Sonication Platform, 1.5 ml		
ChIP-IT® Protein G Magnetic Beads		
Protein G Agarose Columns		
Siliconized Tubes, 1.7 ml		
ChIP-IT® Control qPCR Kit – Human		
ChIP-IT® Control qPCR Kit – Mouse		ChIP-IT® Control qPCR Kit – Rat		ChIP-IT® Control Kit – Human		
ChIP-IT® Control Kit – Mouse		ChIP-IT® Control Kit – Rat		
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		
Dounce Homogenizer		Dounce Homogenizer		

25 rxns
25 rxns
10 rxns
10 rxns
16 rxns
16 rxns
16 rxns
10 rxns
10 libraries
5 rxns
5 rxns96 rxns
16 rxns
16 rxns
25 rxns
25 rxns
50 rxns
110 V
1 platform
25 rxns
30 rxns
25 tubes
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
5 rxns
10 rxns
10 rxns
10 rxns
10 rxns
500 µg
1 ml
15 ml

53008
53009
53032
53035
53084
53040
53046
53029
53041
53031
53030
53018
53084
53216
53016
53024
58002
53051
53080
53014
53039
53036
53026
53027
53028
53010
53011
53012
53015
53019
53020
53021
53017
40401
40415

ChIP-validated Antibodies
For an up-to-date list of ChIP-validated antibodies, please visit www.activemotif.com/chipabs.
Whole Genome Amplification		

FormatCatalog No.

GenoMatrix™ Whole Genome Amplification Kit		

1 kit

58001

Co-Immunoprecipitation		

FormatCatalog No.

Nuclear Complex Co-IP Kit		
Universal Magnetic Co-IP Kit		

50 rxns
25 rxns

54001
54002

www.activemotif.com

21

Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008, USA
E-mail: tech_service@activemotif.com

Toll Free: 877 222 9543
Direct: 760 431 1263
Fax: 760 431 1351

Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
E-mail: eurotech@activemotif.com
Direct: +32 (0)2 653 0001

Germany Free Phone: 0800 181 99 10
France Free Phone: 0800 90 99 79
UK Free Phone: 0800 169 31 47
Fax: +32 (0)2 653 0050

Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan

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

Active Motif China
787 Kangqiao Road
Building 10, Suite 202
Pudong District
Shanghai, 201315, China

Direct: (86)-21-20926090
Hotline: 400-018-8123
E-mail: techchina@activemotif.com

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22