ChIP-IT High Sensitivity®
(version A6)
Catalog No. 53040

Copyright 2016 Active Motif, Inc.

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 kind, either explicit or implied. Information may be changed or updated in this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is proprietary information and protected by the copyright laws of the United States and international
treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2016 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights
reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocol Overview and Time Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Components and Storage
ChIP-IT High Sensitivity Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols – Experimental Set Up
Cell Growth and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Buffer Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Protocols – Preparation of Sheared Chromatin
Section A. Cell Fixation Starting with Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Section B. Chromatin Sonication of Cultured Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section C. Cell Fixation Starting with Fresh or Frozen Tissue . . . . . . . . . . . . . . . . . . . .14
Section D. Chromatin Sonication of Tissue. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Protocols – Chromatin Immunoprecipitation
Section E. Immunoprecipitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Protocols – ChIP DNA Purification
Section F. Reversal of Cross-links and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . 20
Protocols – ChIP DNA Analysis
Section G. Quantitative PCR (qPCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Section H. ChIP-Seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Appendix
Section I. qPCR Primer Design and Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Section J. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

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Please Read Before Starting:
The ChIP-IT High Sensitivity® Kit contains an optimized protocol and buffer
system to ensure the highest level of sensitivity and lowest backgroundwhen working with limited sample amounts or low abundance transcription
factor targets. It is important to follow our protocol and recommendations
throughout the entire manual as many steps may differ from traditional
ChIP protocols. Please pay particular attention to our notes regarding theprocessing of input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction.

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 or
ChIP-chip, insights are possible into gene regulation, gene expression, mechanisms of chromatin
modification and pathway analysis.
However, ChIP can be technically demanding. The method requires high-quality antibodies torecognize the fixed, target-bound proteins of interest, and an efficient means to precipitate theantibody/chromatin complex (usually protein A or G beads). In addition, specialized buffers,
inhibitor cocktails and blocking reagents are required to minimize non-specific enrichment andreduce protein degradation.
Active Motif’s ChIP-IT High Sensitivity® Kit is designed to overcome these obstacles and provide
the highest quality ChIP enriched DNA. The ChIP-IT High Sensitivity protocol can be used withchallenging antibodies that do not give signal with other ChIP methods and is sensitive enoughto detect specific binding of even low abundance transcription factors. One important component of the increased sensitivity are the optimized ChIP buffers which reduce the presence of
non-specific DNA to reduce background levels and allow better enrichment analysis. Additionally,
the kit has demonstrated excellent reproducibility across multiple sample types and immunoprecipitation reactions can be performed from as little as 1,000 cells. The increased sensitivity andreduced background levels of ChIP-IT High Sensitivity are ideal for use in ChIP-Seq. So before investing time and money into expensive downstream genome analysis, ensure that your enriched
DNA is of the highest quality.
The ChIP-IT High Sensitivity Kit contains sufficient reagents to perform 16 chromatin preparationand immunoprecipitation reactions. A chromatin preparation is defined as one 15 cm cell culture
plate or 100 mg tissue sample. We recommend the use of Active Motif’s ChIP-IT® qPCR Analysis
Kit in combination with the ChIP-IT High Sensitivity Kit for complete data analysis. To learn about
available ChIP-IT® Control Kits, control qPCR primer sets, ChIP-Seq validated antibodies, or Active
Motif’s EpiShear™ sonication devices, please visit our website at www.activemotif.com/chip.

product

formatcatalog no.

ChIP-IT High Sensitivity®

16 rxns

53040

ChIP-IT® qPCR Analysis Kit

10 rxns

53029

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1Flow Chart of Process
Cross-link protein to DNA inliving cells with formaldehyde.

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

Break open cells andshear DNA by sonication.

Add primary
antibody of interest.

Elute chromatin fromcolumn and save flow-through.

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

Flow Chart of the ChIP-IT High Sensitivity Assay.
In ChIP-IT High Sensitivity, intact cells are fixed with a specially formulated formaldehyde buffer, which crosslinks and preserves protein/DNA interactions. DNA is then sheared into small fragments using sonication andincubated 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 and purified. ChIP enriched DNA can be used for either gene-specific or wholegenome analysis.

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Kit Performance and Benefits
ChIP-IT High Sensitivity Advantages:• Ideal for low abundance transcription factors or antibodies with low binding affinities• Sensitive enrichment of DNA from as little as 1,000 cells for high abundance target proteins
and as little as 50,000 cells for low abundance transcription factors• Optimized reagents reduce background levels caused by non-specific binding events• Filtration based washes are the easiest wash method available and result in increased
consistency in multi-sample experiments• Highly robust procedure has been validated across multiple sample types with proven performance in both qPCR and ChIP-Seq analysis

ChIP-IT High Sensitivity®
Binding events detected / 1,000 cells

2500
H3K4me3
IgG

2000
1500
1000
500

Negative

GAPDH(AM Catalog #71001)(AM Catalog #71006)

1,000,000

500,000

100,000

10,0001,000

1,000,000

500,000

100,000

10,0001,000

0Figure 1: Immunoprecipitation sensitivity of ChIP-IT High Sensitivity Kit.
MCF-7 chromatin was prepared according to the manual using between 100,000 to 4 million cells per chromatin preparation. The immunoprecipitation reaction was then performed using the indicated number of cellswith Active Motif’s Histone H3K4me3 antibody (Catalog No. 39915) and a negative control IgG. Following enrichment, qPCR was performed using the ChIP-IT qPCR Analysis Kit (Catalog No. 53029) in order to normalize
the data and allow for direct comparison of the results. The ChIP-IT High Sensitivity Kit shows strong enrichment of H3K4me3 DNA binding from as little as 1,000 cells, while little to no non-specific binding is detectedwith the negative control IgG. Untr12 is a gene desert on chromosome 12 that should not show any H3K4me3
enrichment, while GAPDH is an actively transcribed gene that is associated with the presence of H3K4me3.
Data is expressed as binding events detected per 1,000 cells which represents the average of the raw data
triplicates adjusted for the amount of chromatin in the reaction, the resuspension volume and the primer
efficiency. This calculation provides consistency in data analysis and allows direct comparison across samples
and experiments. To convert this scale to the percent of ChIP input recovered, divide the values by 1,000.
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3Comparison of ChIP Kits Targeting Low Abundance Transcription Factors
Binding events detected / 1,000 cells

35High background &non-specific binding

30
25
20

NCOA2
IgG

15
10
5
0

Negative

ESRRA

Negative

ChIP-IT HS

ESRRA

Negative

Competitor M

ESRRA

Negative

Competitor I

ESRRA

Competitor D

Figure 2: ChIP-IT High Sensitivity can detect low abundance protein targets.
MCF-7 chromatin was prepared according to each manufacturer’s recommendations for their ChIP assay
from 1.5 x 106 cells. ChIP was performed with the optimal amount of chromatin as suggested by the protocol
for each manufacturer using an antibody for the low abundance nuclear co-activator 2 (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 normalize the data for chromatin amounts and ChIP volume to allow for directcomparison of the competitor kit results. NCOA2 is considered a difficult antibody for ChIP, yet using ChIP-IT
High Sensitivity, NCOA2 binding was detected at the estrogen-related receptor alpha (ESRRA) promoter andenrichment was approximately 20-fold higher than the negative control primer set and the IgG. NCOA2 waseither not enriched at all (Competitors M & I) or the enrichment was non-specific with high background visible
in the negative primer set and IgG (Competitor D). The data represents triplicate values expressed as Binding
events detected per 1,000 cells. To convert this scale to the percent of ChIP input recovered, divide thevalues by 1,000.

Protocol Overview and Time Table
Required Time
Cell or Tissue Fixation and Lysis

1.5 hours

Chromatin Sonication

15 minutes per sample

Assessment of Chromatin Size*

4.5 hours for cell culture
Overnight for tissue

Immunoprecipitation

Overnight incubation

Binding to Protein G agarose Beads

3 hours

Wash Immune Complexes

20 minutes

Reversal of Cross-links

2.5 hours

DNA Purification

15 minutes

qPCR Analysis

2 hours* The protocol varies between cell culture and tissue samples.
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4Kit Components and Storage
Please store each component at the temperature indicated in the table below. Do not re-freeze
the Protein G Agarose Beads. Once thawed, Protein G beads should be stored at 4°C.
Reagents

Quantity

Storage

RNase A (10 µg/µl)

40 µl

-20°C

Proteinase K (10 µg/µl)

200 µl-20°C

Blocker

100 µl-20°C

5 M NaCl

400 µlRT

100 mM PMSF

500 µl-20°C

Protease Inhibitor Cocktail (PIC)

500 µl-20°C

Precipitation Buffer

1.5 ml-20°C

Carrier

35 µl

-20°C

TE pH 8.0

2 x 1.5 ml

RTDetergent

25 ml

RT

10X PBS

120 ml-20°C

Fixation Buffer

2 x 1.5 ml

4°C

Stop Solution

20 ml

RT

Chromatin Prep Buffer

85 ml

RT

ChIP Filtration Columns

16 ea

RT

ChIP Buffer

35 ml

RT

Wash Buffer AM1

100 mlRT

Elution Buffer AM4

2 x 1.5 ml

RTProtein G Agarose Beads*

500 µl4°C

DNA Purification Binding Buffer

50 ml

RT

3 M Sodium Acetate

500 µlRT

DNA Purification Wash Buffer**

10 ml

RT

DNA Purification Elution Buffer

5 ml

RT

DNA Purification Columns

16 ea

RT

* The Protein G Agarose Beads are shipped on dry ice and can be stored frozen until their firstuse. Once thawed, the Protein G beads should not be re-frozen by the customer. Protein G
Agarose Beads should be stored at 4ºC.
**Requires the addition of ethanol before use.
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5Additional materials required• A ChIP-validated antibody directed against the protein of interest• Dounce homogenizer with a small clearance pestle (e.g. Active Motif Catalog Nos. 40401& 40415) with the tight-fitting “A” pestle). Use of a homogenizer is necessary for shearing
chromatin.
• 37% formaldehyde solution with 10-15% methyl alcohol to prevent polymerization (e.g.
Sigma Aldrich Catalog No. 252549). Do not use paraformaldehyde.
• For tissue preparations you will need phenol, saturated (DNA Purification, Molecular Biology
Grade, Amresco Catalog No. 0945)• For tissue preparations you will need chloroform/isoamyl alcohol (24:1) (DNA Purification,
Molecular Biology Grade)• 100% ethanol (absolute)• 70% ethanol• DNase-free H2O• Rocking platform for culture plates• 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• Thermocycler• 15 and 50 ml conical tubes• Spectrophotometer for DNA quantitation• Pipettors and tips (filter tips are recommended)• Sonicator (e.g. Active Motif’s EpiShear™ Sonicator with a 1/8” probe (Catalog No. 53051))• Agarose gel electrophoresis apparatus• Razor blades (for tissue preparations)• Hand-held homogenizer for tissue preparations (e.g. Biospec Products Tissue-Tearor)• Cell scraper (rubber policeman)• (Optional) ChIP-IT® qPCR Analysis Kit (Catalog No. 53029)• (Optional) Gene-specific qPCR primer pairs for enrichment analysis; see Appendix Section I• (Optional) SYBR Green qPCR master mix (Bio-Rad Catalog No. 170-8882)

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Protocols – Experimental Set Up
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Cell Growth Recommendations
When planning an experiment, calculate the number of chromatin preparations you will requireand determine the number of ChIP reactions you plan to perform on each chromatin preparation. Be sure to include the appropriate positive and negative control ChIP reactions in yourcalculations. Also, note that if you wish to analyze the effect of particular compounds or culturingconditions on transcription factor/DNA interactions, you should prepare chromatin from control(untreated) cells as a reference sample. The minimum recommended number of cells that should
be used for the preparation of chromatin is 100,000 cells.
24-wellplate

12-wellplate

6-well
plate

60 mm
dish

100 mmdish

150 mmdish

Seeding Density

0.05 x 106

0.1 x 106

0.3 x 106

0.8 x 106

2.2 x 106

5.0 x 106

Cells at 70-80% Confluency*

0.15 x 106

0.3 x 106

0.9 x 106

2.4 x 106

6.6 x 106

15.0 x 106

Growth Medium Volume

1 ml

2 ml

3 ml

5 ml

10 ml

20 ml

Cell Fixative Solution

100 µl200 µl300 µl500 µl

1 ml

2 ml

Stop Solution

55 µl

110 µl165 µl

275 µl

550 µl1.1 mlPBS Wash Buffer(used per wash)

500 µl500 µl

1 ml

2 ml

5 ml

10 ml

Chromatin Prep Buffer

500 µl500 µl

1 ml

2 ml

5 ml

5 ml

ChIP Buffer

500 µl500 µl500 µl500 µl500 µl500 µl* The number of cells on a confluent plate or dish will vary with cell type. For this table, HeLa
cells were used. Please adjust as needed based on your particular cell type.
** Please refer to the descriptions below for complete details on buffer preparations

Buffer Preparation
Complete Cell Fixation Solution
Buffer should be prepared fresh before each experiment. For every 20 ml of cell growth medium
used, prepare 2.5 ml of Complete Cell Fixation Solution by adding 180 µl Fixation Buffer to 1.57
ml sterile water in a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves
and lab coat), add 750 µl 37% formaldehyde to the tube and vortex to mix. Use 1/10 growth
medium volume per plate. Complete cell fixation solution can be added to the growth medium inthe presence or absence of serum.
Complete Tissue Fixation Solution
Buffer should be prepared fresh before each experiment. Prepare 10 ml of Tissue Fixation
Solution for each tissue sample to be processed by adding 1 ml 10X PBS to 8.7 ml sterile water in
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7

a 15 ml conical tube. Using appropriate precautions (i.e. safety glasses, gloves and lab coat), add
280 µl 37% formaldehyde to the tube and vortex to mix.
Stop Solution
Is provided ready to use. Use 1/20 media volume per cell culture plate or 515 µl per 10 ml Complete Tissue Fixation Solution.
PBS Wash Buffer
Prepare 25 ml PBS Wash Buffer for every 15 cm plate or tissue sample. To a 50 ml conical tube
add 21.25 ml sterile water, 2.5 ml 10X PBS and 1.25 ml Detergent. Mix by inverting. Place PBS
Wash Buffer on ice to chill. PBS Wash Buffer can be prepared in large quantities and stored at 4ºC
for 6 months.
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.
Chromatin Prep Buffer
Is supplied ready to use.
ChIP Buffer
Is supplied ready to use.
DNA Purification Wash Buffer
The DNA Purification Wash Buffer requires the addition of ethanol before use. The final concentration of ethanol should be 80%. Add 40 ml of fresh 100% ethanol to the DNA Purification Wash
Buffer bottle. Invert repeatedly. The buffer can be stored at room temperature after the additionof ethanol. The ethanol only needs to be added before the first use, after that the Wash Buffer isready for use.
3M Sodium Acetate
It is important to check the sodium acetate before use to ensure that the salts have notprecipitated out of solution. Once the sodium acetate is in solution it should be stored at room
temperature.
Protein G Agarose Beads
The supplied agarose beads require washing prior to use. Follow the instructions in the manual
to wash the beads for use in the ChIP reactions. There is no need to pre-block the beads or
pre-clear the sample. 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 mm from the end of a pipet tip prior to pipetting to prevent the tip frombecoming 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.
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8

Recommendations
ChIP-validated Antibody
We recommend using 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-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 Shearing Tips
We suggest using a probe sonicator (i.e. Active Motif’s EpiShear Probe Sonicator) which employs
a direct sonication method to prepare chromatin for use in the ChIP-IT High Sensitivity Kit. Indirect
sonication systems may require longer sonication times to achieve optimal chromatin shearing.
ChIP experiments usually require chromatin that has been sheared to a size of 200-1200 bp. In
general, shearing efficiency is improved through the use of a small shearing volume and a Vbottom tube rather than a round-bottom tube. Also, note that shearing is inefficient if the chromatin
sample becomes emulsified with air bubbles. To determine the appropriate shearing level for yoursample, set up a “practice” tube containing only ChIP Buffer. Slowly increase the sonication amplitude until foaming starts to occur. Reduce the amplitude setting down slightly and mark this as thehighest possible intensity to use without foaming. If a chromatin preparation becomes emulsified
inadvertently, discontinue shearing and centrifuge the sample at maximum speed for 4 minutes at
4ºC in a microcentrifuge to remove trapped air. Finally, to prevent overheating and denaturationof chromatin, samples should be kept on ice as much as possible during shearing, and shearing
should be performed discontinuously (i.e. sonicate for 20 seconds, then place on ice/water for
30 seconds, sonicate again for 20 seconds, etc.). If possible, shear while on ice to help regulate
sample temperature.
Chromatin Quantity
It is recommended to use 10-30 µg chromatin per IP reaction (1.5-4.5 million cells). However, if
chromatin is in limited supply lower amounts may be used due to the high quality of the DNA
obtained from the ChIP-IT High Sensitivity Kit. We recommend at least 1,000 cells per IP for highabundance targets and 50,000 cells per IP for low abundance target (assume 1.5 x 106 cells
will yield 10 µg chromatin). If ChIP-Seq will be performed following enrichment, please read therecommendations listed in Section H on page 22 prior to starting the protocol.
Safety Precautions
Formaldehyde and PMSF are highly toxic chemicals. Appropriate safety precautions (i.e. safety
glasses, gloves and lab coat) should be used. Also, formaldehyde is highly toxic by inhalation andshould be used only in a ventilated hood. Finally, chromatin sonication should be performed in abiosafety hood if the chromatin is extracted from biohazardous or infectious materials.
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Protocols – Preparation of Sheared Chromatin
Section A: Cell Fixation Starting with Cultured Cells
This protocol describes cell fixation and chromatin preparation from one 15 cm plate (approximately 1.5 x 107 cells). We recommend using 20 ml growth medium per 15 cm plate. Pleaserefer to page 7 for information on scaling the protocol for use with other amounts of cells. The
minimum cell number to be used for the preparation of chromatin is 100,000 cells.
1.

Prepare 15 cm plates for each cell line to be tested. Grow the cells to 70-80% confluency.
Stimulate cells as desired to activate the pathway of interest.

2.

Freshly prepare Complete Cell Fixation Solution for each 15 cm plate. The volumes listed
below are enough to process one 15 cm plate. Please refer to the chart on page 7 to scale
the solution volumes.

3.

To fix cells, add 1/10 growth medium volume of freshly prepared Complete Cell Fixative
Solution to the existing culture media for the cells (e.g. 20 ml growth medium would get
2 ml Complete Cell Fixation Solution). Shake gently at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 1/20 media volume of Stop Solution to the existing
culture media for the cells (e.g. 20 ml growth medium would get 1.1 ml Stop Solution). Swirlto mix and incubate at room temperature for 5 minutes.

5.

Following the incubation, hold the plate at an angle and using a rubber policeman scrape
cells down to collect them at the bottom edge of the plate. Use a pipette to transfer thecells to a 50 ml conical tube on ice.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet(s) in 10 ml ice-cold PBS Wash
Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets
may be stored at -80°C at this stage).

9.

Resuspend each pellet(s) in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF. Pipet up and down to mix.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellets individually to a chilled dounce homogenizer on ice.
Use the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Transfer thecontents to a new 15 ml conical tube and centrifuge for 3 minutes at 1,250 x g at 4°C.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce andlook at it under a phase contrast microscope using a hemocytometer to verify that thenuclei have been released. It is often helpful to look at the cells before and after the lysis
step as this makes it easier to identify the nuclei versus whole cells. Intact cells should havea dark central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells,
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10the nuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed,
then dounce on ice with an additional 10 strokes, or until the cells are lysed.
12. Remove the supernatant and discard. Resuspend each pellet in 500 µl ChIP Buffer supplemented with 5 µl PIC and 5 µl 100 mM PMSF. Transfer the contents to a new 2 ml microcentrifuge tube.
13. Incubate on ice for 10 minutes. Proceed to Step B: Chromatin Sonication of Cultured Cells.

Section B. Chromatin Sonication of Cultured Cells
The section below describes the fragmentation of chromatin using sonication. Sonication results
may vary depending on cell type and sonication device being used. This protocol has been validated using Active Motif’s EpiShear™ Probe Sonicator with samples on ice and the probe height
maintained between samples to ensure consistency in processing. We do not recommend sonication of samples containing less than 100,000 cells and/or 350 µl volume.
The Chromatin Prep Buffer has been optimized for immunoprecipitation performance, however,
due to its unique composition optimization of sonication conditions may be required. To maintainthe high sensitivity of the assay, we recommend using our buffer system and altering the sonication time and/or amplitude of your sonication system to achieve the desired fragmentation(e.g. some systems may require as much as a three-fold increase in sonication time to improve
chromatin shearing). Please pay particular attention to our protocol regarding the processing of
input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as
many steps may differ from traditional ChIP protocols and failure to follow the outlined procedure
may lead to artifacts in the gel images as shown in Figure 3 on page. 13
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the cell typebeing used (see Recommendations on page 9). A recommended starting range for cultured
cells is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total sonication“on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to
the immunoprecipitation step.

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11Input Preparation5.

To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
1 µl RNAse A. Cap the PCR tubes and vortex to mix

6.

Incubate in a thermocycler at 37°C for 30 minutes.

7.

Add 2 µl Proteinase K to each tube and vortex. Incubate tubes in a thermocycler at 55°C for
30 minutes and then increase the temperature to 80°C for 2 hours.

8.

Transfer each chromatin input to a 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 750 µl absolute ethanol. Vortex to mix and chill at -80°C for 30 minutes
to overnight.

9.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 15 minutes.

10. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
11.

Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.

12. When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.
13. Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 14. Store the remaining Input DNA at -20°C.
14. Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping thetemperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

Note:

Chromatin prepared using the ChIP-IT High Sensitivity protocol may look different on an agarose gel compared to chromatin prepared using traditional ChIP
methods. However, this will not affect the sensitivity of the assay or increase
background signal. Please follow the protocol as listed above for preparing
Input DNA. Use of an alternative reverse cross-linking method or omitting the 20
minute incubation at 100°C in NaCl is not recommended as this will cause artifacts that make the DNA appear larger. As long as the chromatin falls within therecommended 200-1200 bp range, proceed with the ChIP reaction. If fragments
do not fall within this range sonication conditions should be further optimized.

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1215. If chromatin preparations were successful, the aliquots stored at -80°C from Section B, Step
4 can be used to perform the ChIP reactions in Section E.

Figure 3: Validation of chromatin shearing efficiency following reversal of cross-links at 80°C for 2 hours.
Chromatin preparations of MCF-7 cells were fixed and sonicated using the EpiShear™ Probe Sonicator. Input
DNA was prepared in duplicate according to Section B, Steps 5-13 in the manual. In Sample 1, Step 14 was
not performed and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel without receiving theaddition of NaCl and incubation at 100°C. The omission of Step 14 has caused a buffer artifact that makes
the DNA appear larger on a gel. The duplicate sample, Sample 2, was processed according to the manual
instructions and included the addition of NaCl and incubation at 100°C as stated in Step 14. Analysis of 500 ng
of this input DNA on a 1.5% agarose gel shows the expected fragmentation between 200-1200 bp. The difference in DNA sizing on the gel between the two samples illustrates the importance of following the protocol
recommendations regarding the processing of input chromatin for agarose gel analysis prior to chromatin immunoprecipitation. Omission of key steps can lead to inaccurate analysis of chromatin shearing efficiency. If
the protocol steps were followed and the DNA fragments fall outside of the recommended range, sonication
conditions should be further optimized.

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13Section C: Cell Fixation Starting with Fresh or Frozen Tissue
This protocol describes cell fixation and chromatin preparation from 100-400 mg fresh or frozen
animal tissue. If performing chromatin preparation on multiple tissue samples, we recommend
completing Steps 1-7 for each sample before processing the next sample.
1.

For tissue fixation, transfer 10 ml Complete Tissue Fixation Solution (see Buffer Preparation
on page 7) to a 60 mm petri dish. Place the dish on ice.

2.

Add 100-400 mg fresh or frozen tissue sample to the petri dish and ensure that the sample
is fully immersed. Cut the tissue sample into small pieces (approximately 1 mm cubes) usinga razor blade.

3.

Transfer the sample plus the Complete Tissue Fixation Solution to a 15 ml conical tube androtate at room temperature for 15 minutes.

4.

Stop the fixation reaction by adding 515 µl Stop Solution to the conical tube and rotate atroom temperature for 5 minutes.

5.

Place the conical tube on ice and homogenize the contents with a hand-held tissue homogenizer set at 30,000 rpm for 45 seconds.

6.

Pellet the cells from step 5 by centrifugation for 3 minutes at 1,250 x g at 4°C.

7.

Remove the supernatant and discard. Resuspend the pellet in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Keep samples ice-cold for the remainder of the procedure.

8.

Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. Wash thepellet(s) a second time in 10 ml ice-cold PBS Wash Buffer by pipetting up and down. Centrifuge for 3 minutes at 1,250 x g at 4°C. Remove the supernatant and discard. (Cell pellets
may be stored at -80°C at this stage).

9.

Resuspend each pellet in 5 ml Chromatin Prep Buffer supplemented with 5 µl PIC and5 µl 100 mM PMSF.

10. Incubate on ice for 10 minutes.
11.

Transfer the resuspended pellet(s) individually to a chilled dounce homogenizer on ice. Use
the tight fitting pestle (Type A) to homogenize the sample for 30 strokes. Once finished,transfer the contents to a new 15 ml conical tube.
Monitor Cell Lysis: To ensure cell lysis, take 10 µl of the cell lysate from the dounce andlook at it under a phase contrast microscope using a hemocytometer to verify that thenuclei have been released. It is often helpful to look at the cells before and after the lysis
step as this makes it easier to identify the nuclei versus whole cells. Intact cells should havea dark central region (nucleus) surrounded by a halo of less dense cytoplasm. In lysed cells,
the nuclei will appear as dots surrounded by asymmetric debris. If the cells are not lysed,
then dounce on ice with an additional 10 strokes, or until the cells are lysed.

12. Centrifuge for 3 minutes at 1,250 x g at 4°C.
13. Remove the supernatant and discard. Resuspend each pellet in 500 µl - 1 ml ChIP Buffer
supplemented with PIC and 100 mM PMSF. (For 500 µl add 5 µl PIC and 5 µl PMSF. For 1 ml
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14

add 10 µl PIC and 10 µl PMSF.) Transfer the contents to a new 2 ml microcentrifuge tube.
14. Incubate on ice for 10 minutes. Proceed to Section D: Chromatin Sonication of Tissue.

Section D. Chromatin Sonication of Tissue
The section below describes the fragmentation of chromatin using sonication. Due to theincreased concentration of protein and cellular debris present in animal tissue, we recommend
following this protocol for the preparation of chromatin and input DNA from tissue. Sonication
results may vary depending on tissue type and sonication device being used. This protocol has
been validated using Active Motifs EpiShear™ Probe Sonicator with samples on ice and the probeheight maintained between samples to ensure consistency in processing. We do not recommend
sonication of samples containing less than 50 mg tissue and/or 350 µl volume.
The Chromatin Prep Buffer has been optimized for immunoprecipitation performance, however,
due to its unique composition optimization of sonication conditions may be required. To maintainthe high sensitivity of the assay, we recommend using our buffer system and altering the sonication time and/or amplitude of your sonication system to achieve the desired fragmentation(e.g. some systems may require as much as a three-fold increase in sonication time to improve
chromatin shearing). Please pay particular attention to our protocol regarding the processing of
input chromatin for agarose gel analysis prior to the chromatin immunoprecipitation reaction as
many steps may differ from traditional ChIP protocols and failure to follow the outlined procedure
may lead to artifacts in the gel images as shown in Figure 4 on page 17.
1.

Place the 2 ml microcentrifuge tube containing the chromatin into the tube cooler or packed
ice. Open cap and submerge the microtip into the liquid until the microtip is approximately
5 mm from the bottom of the tube. Sonicate according to optimized settings for the tissue
type being used (see Recommendations on page 9). A recommended starting range fortissue samples is: 25% amplitude, pulse for 30 seconds on and 30 seconds off for a total
sonication “on” time of 10 minutes (or 20 minutes elapsed time).

2.

Spin tubes at 4°C in a microcentrifuge at maximum speed for 2 minutes to pellet the cellulardebris.

3.

Transfer 25 µl of each chromatin preparation into a 250 µl PCR tube for analysis of shearing
efficiency and chromatin quantification. This sample will be used to generate the Input DNA.

4.

Aliquot the remainder of each chromatin preparation into 1.5 ml microcentrifuge tubes. We
recommend making aliquots of 150 µl volume and storing at -80°C.
Note:

The size of the chromatin sonication should be verified before proceeding to
the immunoprecipitation step.

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15Input Preparation5.

To each 25 µl chromatin preparation from Step 3 above, add 175 µl TE pH 8.0 and
2 µl RNAse A. Cap the PCR tubes and vortex to mix.

6.

Incubate in a thermocycler at 37°C for 1 hour.

7.

Add 5 µl Proteinase K to each tube, vortex and incubate in a thermocycler at 37°C for 3
hours.

8.

Add 10 µl 5 M NaCl, vortex and incubate at 65°C for 6-16 hours to reverse cross-links.

9.

Remove tubes from the thermocycler and add 250 µl phenol and 125 µl chloroform:isoamyl
alcohol (24:1). Vortex vigorously and spin tubes in a room temperature microcentrifuge at
maximum speed for 2 minutes.

10. Transfer each upper aqueous layer to a new 1.5 ml microcentrifuge tube and add 250 µl
chloroform:isoamyl alcohol (24:1). Vortex vigorously and spin tubes in a room temperature
microcentrifuge at maximum speed for 2 minutes.
11.

Transfer the upper aqueous layer to a new 1.5 ml microcentrifuge tube. Add 83 µl Precipitation Buffer, 2 µl Carrier and 900 µl absolute ethanol. Vortex to mix and chill at -80°C for 30
minutes to overnight.

12. Spin at 4°C in a microcentrifuge at maximum speed for 15 minutes.
13. Carefully remove the supernatant taking care not to disturb the pellet. Wash the pellet with
500 µl 70% ethanol and spin at 4°C in a microcentrifuge at maximum speed for 5 minutes.
14. Carefully remove the supernatant taking care not to disturb the pellet. Remove residual
ethanol with a pipet tip. Leave the tubes uncapped and air dry for 10-15 minutes.
15. When the pellets are dry, add 25 µl DNA Purification Elution Buffer to each tube. Incubate atroom temperature for 10 minutes. Then vortex to ensure the pellet is completely resuspended. This solution contains your Input DNA.
16. Read the absorbance of each sample on a NanoDrop or other spectrophotometer at
260 nm to determine the DNA concentration of each chromatin preparation. Set aside
500 ng of DNA for analysis as described in Step 17. Store the remaining Input DNA at -20°C.
17.

Analyze each chromatin preparation on an agarose gel by following the instructions below.
a.

Prepare 500 mM NaCl by adding 2 µl 5M NaCl to 18 µl sterile water. Vortex to mix.

b.

Transfer 500 ng of Input DNA to a 250 µl PCR tube and add 1 µl 500 mM NaCl. Adjust
the final volume to between 10 µl with sterile water if needed.

c.

Heat samples in a thermocycler at 100°C for 20 minutes followed by ramping thetemperature down to 50°C.

d.

Remove tubes from the thermocycler and incubate at room temperature for 5 minutes.

e.

Add gel loading buffer to each sample and run on a 1.5% agarose gel. Include 100 bp
and 1 kb DNA ladders to analyze chromatin size. DNA should appear as a smear anywhere between 200-1200 bp.

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16

Note:

Chromatin prepared using the ChIP-IT High Sensitivity protocol may look different on an agarose gel than chromatin prepared using traditional ChIP methods.
However, this will not affect the sensitivity of the assay or increase backgroundsignal. Please follow the protocol as listed above for preparing Input DNA. Useof an alternative reverse cross-linking method or omitting the 20 minute incubation at 100°C in NaCl is not recommended as this will cause artifacts that makethe DNA appear larger. As long as the chromatin falls within the recommended
200-1200 bp range, proceed with the ChIP reaction. If fragments do not fallwithin this range sonication conditions should be further optimized.

18. If chromatin preparations were successful, the aliquots stored at -80°C from Section D,
Step 4 can be used to perform the ChIP reactions in Section E.

Figure 4: Validation of chromatin shearing efficiency following reversal of cross-links overnight at 65°C.
Chromatin preparations were fixed and sonicated using the EpiShear™ Probe Sonicator. Input DNA was prepared in duplicate according to Section D, Steps 5-16 in the manual. In Sample 1, Step 17 was not performed
and 500 ng of input DNA was loaded directly onto a 1.5% agarose gel without receiving the addition of NaCland incubation at 100°C. The omission of Step 17 has caused a buffer artifact that makes the DNA appear
larger on a gel. The duplicate sample, Sample 2, was processed according to the manual instructions and included the addition of NaCl and incubation at 100°C as stated in Step 17. Analysis of 500 ng of this input DNA
on a 1.5% agarose gel shows the expected fragmentation between 200-1200 bp. The difference in DNA sizingon the gel between the two samples illustrates the importance of following the protocol recommendations
regarding the processing of input chromatin for agarose gel analysis prior to chromatin immunoprecipitation.
Omission of key steps can lead to inaccurate analysis of chromatin shearing efficiency. If the protocol steps
were followed and the DNA fragments fall outside of the recommended range, sonication conditions should
be further optimized.

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17

Protocols – Chromatin Immunoprecipitation
Section E. Immunoprecipitation
Successful chromatin immunoprecipitation depends on the quality of the ChIP antibody and theabundance of the target protein. For high abundance proteins as little as 1,000 cells can be used
for each immunoprecipitation reaction. For lower abundance proteins as little as 50,000 cells canbe used for each immunoprecipitation reaction. (1.5 x 106 cells is equivalent to 10 µg chromatin)
1.

Thaw sonicated chromatin on ice. Spin chromatin at 4°C in a microcentrifuge at maximum
speed for 2 minutes.

2.

Set up the ChIP reactions by adding the components in the order shown in Table 1 below
to 1.5 ml microcentrifuge tubes. Be sure to use the DNA concentration that was determinedfor your sonicated chromatin sample to calculate the volume to use. We recommend using
10-30 µg chromatin (1.5 -4.5 million cells) per ChIP reaction, although less can be used if
chromatin yields are limiting.

3.

In a 1.5 ml microcentrifuge tube prepare the antibodies to be used in the ChIP reactions.
Use a separate tube for each antibody. To the tube add 5 µl Blocker and 4 µg ChIP antibody.
(Antibody volume should not exceed 30 µl per reaction). Incubate Antibody/Blocker mix for 1
minute at room temperature and then add to the ChIP reactions.
Table 1

Reagent

1 reaction

Sheared Chromatin (10 - 30 µg)

X µl

ChIP Buffer

adjust up to 200 µl

Protease Inhibitor Cocktail (PIC)

5 µl

Antibody/Blocker mix (from Step 3)

not to exceed 35 µl

Maximum Volume Allowed

240 µl4.

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

5.

The Protein G agarose beads require washing before use. Transfer 30 µl Protein G agarose
beads for each IP reaction to a 1.5 ml microcentrifuge tube. Add an equal volume of TE,
pH 8.0 and invert to mix. Spin at 1250 x g in a microcentrifuge for 1 minute. Remove thesupernatant equivalent to the volume of TE added to the agarose beads.
Note:

6.

Before pipetting the Protein G agarose beads, they should be fully resuspended
by inverting the tube. When pipetting the beads, cut 2 mm from the end of apipet tip to prevent the tip from becoming clogged.

Wash the beads a second time with the same volume of TE, pH 8.0. Invert to mix. Spin
at 1250 x g for 1 minute in a microcentrifuge. Remove the supernatant equivalent to the
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18

volume of TE added to the agarose beads. The beads are now ready to use.
7.

Spin the ChIP reactions at 1250 x g for 1 minute to collect liquid from the inside of the cap.

8.

Using a cut pipet tip, add 30 µl washed Protein G agarose beads to each immunoprecipitation reaction. Cap tubes and incubate on an end-to-end rotator at 4°C for 3 hours.

9.

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 (see Figure 5 below).

10. Remove ChIP reactions from rotator and spin at 1250 x g for 1 minute to collect liquid frominside of the cap.
11.

Add 600 µl ChIP Buffer to each ChIP reaction then transfer the entire reaction (including theprotein G agarose beads) to its labeled column. Allow flow-through to occur by gravity.

12. During the gravity flow, transfer 100 µl per ChIP reaction of Elution Buffer AM4 to a 1.5 ml
microcentrifuge tube and allow to pre-warm at 37°C during the wash steps.
13. Wash each column with 900 µl Wash Buffer AM1. Let stand for 3 min.
14. Repeat Step 13 four more times for a total of five washes.
15. Transfer columns to a new 1.5 ml microcentrifuge tube and spin in a room temperature
microcentrifuge at 1250 x g for 3 minutes to remove residual Wash Buffer.
16. Following the spin, transfer the ChIP Filtration Columns to new 1.5 ml microcentrifuge tubes.
Add 50 µl 37°C Elution Buffer AM4 to each column. Incubate at room temperature for5 minutes. Spin in a room temperature microcentrifuge at 1250 x g for 3 minutes.
17.

With columns remaining in the same microcentrifuge tube, add another 50 µl 37°C Elution
Buffer AM4 to each column. Incubate at room temperature for 5 minutes and spin in a room
temperature microcentrifuge at 1250 x g for 3 minutes.

18. Discard the ChIP Filtration Columns. The flow-through (~100 µl volume) contains the ChIP
DNA. Proceed to Section F: DNA purification.

Figure 5: Using the ChIP Filtration Columns.
Remove the tab from the bottom of the ChIP Filtration Columns and place columns in an empty 1 ml pipet tip
box to perform the wash steps.
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19

Protocols – ChIP DNA Purification
Section F. Reversal of Cross-links and DNA Purification
1.

Transfer each eluted ChIP DNA to a 250 µl PCR tube and add 2 µl Proteinase K. Vortex tomix and heat in a thermocycler at 55°C for 30 minutes and then increase the temperature to
80°C for 2 hours.

2.

Transfer the DNA to a 1.5 ml microcentrifuge tube and add 5 volumes (500 µl) DNA Purification Binding Buffer to each tube and vortex to mix. Adjust the pH with 5 µl 3M Sodium
Acetate. The sample should be bright yellow in color to indicate a proper pH. If your sample
is not bright yellow, please refer to the Troubleshooting guide in the Appendix on page 26
for details to adjust pH prior to loading the sample into the purification column.

3.

For each sample, place a DNA purification column (AM #103928) in the collection tube andadd each pH adjusted sample to its own column. Close the cap on each column, place themwith the collection tubes in a microcentrifuge and spin them at 14,000 rpm for 1 minute.

4.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

5.

Prepare DNA Purification Wash Buffer (AM #103497) before the first use. Follow the instruction on page 8 for the addition of ethanol prior to using the solution. Add 750 µl DNA
Purification Wash Buffer to each column and cap the column.

6.

Spin at 14,000 rpm for 1 minute in a microcentrifuge.

7.

Remove the column from the collection tube, then remove and discard the flow throughfrom the collection tube. Return the column to the collection tube.

8.

With the column cap open, spin at 14,000 rpm for 2 minutes in a microcentrifuge to remove
any residual Wash Buffer from the column.

9.

Transfer the column to a clean microcentrifuge tube. Depending on the method to be used
for downstream analysis of the ChIP enriched DNA, use the appropriate elution volume. For
ChIP-Seq or ChIP-chip applications, we recommend pre-warming the required elution buffer
volume at 37°C for 5 minutes prior to use.
a.

For qPCR analysis: add 100 µl of DNA Purification Elution Buffer (AM #103498) to thecenter of the column matrix and incubate for 1 minute at room temperature. Spin at
14,000 rpm for 1 minute in a microcentrifuge. Add an additional 100 µl DNA Purification
Elution Buffer to the column and incubate for 1 minute at room temperature. Spin at
14,000 rpm for 1 minute in a microcentrifuge. Total elution volume is 200 µl.

b.

For ChIP-Seq or ChIP-chip analysis: add 36 µl of 37°C DNA Purification Elution Buffer(AM #103498) to the center of the column matrix and incubate for 1 minute at room
temperature. Spin at 14,000 rpm for 1 minute in a microcentrifuge.

10. Discard column. Purified DNA may be stored at -20°C for future use.

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20

Protocols – ChIP DNA Analysis
Section G. Quantitative PCR (qPCR)
ChIP DNA can be analyzed on a gene-specific basis using quantitative PCR (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 of interest. Active Motif recommends the use of its ChIP-IT® qPCR Analysis
Kit (Catalog No. 53029) for the analysis of qPCR data. The ChIP-IT qPCR Analysis Kit contains
positive and negative control primer pairs, standard curve DNA, standard curve primers and aqPCR Analysis spreadsheet to perform the 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. The
ChIP-IT qPCR Analysis Kit provides consistency in data analysis and allows direct comparison
across samples and experiments. If not using the ChIP-IT qPCR Analysis Kit, qPCR data normalization and graphing can be done using the methods described in Section I.
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.
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.

If using the ChIP-IT qPCR Analysis Kit, please refer to the product manual for analysis
instructions. Otherwise, follow the recommendations in Section I.
Notes:The polymerase chain reaction (PCR) process for amplifying nucleic acid iscovered by U.S. Patent Nos. 4,683,195 and 4,683,202 assigned to Hoffmann-La
Roche. Patents pending in other countries.

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21Section H. ChIP-Seq
The ChIP-IT High Sensitivity Kit has been extensively validated for whole genome analysis using
ChIP-Seq. This process involves the preparation of libraries from ChIP DNA by the addition ofadapter sequences to the ends of the DNA fragments. The library is then PCR amplified andvalidated prior to sequencing.
Commercial kits are available to prepare the ChIP DNA libraries for sequencing. Select a library
preparation kit compatible with the sequencing platform to be used.
A. General Recommendations• We recommend using 30 µg chromatin for the ChIP Reactions, although less may be used if
chromatin amounts are limiting or you are enriching for a highly abundant histone protein• Library generation usually requires 10 ng of ChIP enriched DNA. However, by using the
ChIP-IT High Sensitivity Kit the enriched DNA is of such a high quality that lower amounts
can be used when working with low abundance transcription factors. (See B. ChIP-Seq
Requirements below for more details)• Sequence library yields should be in the range of 1-2 micrograms• 30 million sequencing reads is sufficient for most transcription factor and histone modifications• 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”
and also 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(from Section B Step 12 for cultured cell samples or Section D Step 15 for tissue samples) forlibrary generation.
B. ChIP-Seq Requirements
It is commonly recommended that library generation of ChIP DNA requires 10 ng of ChIP enriched
DNA. Obtaining 10 ng of ChIP DNA should be attainable when using good antibodies against
abundant histone modifications. However, 10 ng is an unrealistic number for a sequence specific
DNA binding transcription factor. Using the ChIP-IT High Sensitivity Kit, we have routinely generated high quality libraries from sub-nanogram levels of ChIP DNA due to the high quality of
the DNA obtained. 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.

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22

C. qPCR prior to ChIP-Seq
To perform qPCR for verification of the quality of the enriched ChIP DNA, follow the instructions
for qPCR in Section G on page 21 with the following modification. The ChIP DNA will need to bediluted prior to use in qPCR due to the reduced elution volume. Dilute 6 µl of ChIP DNA in 94 µl
DNA Purification Elution Buffer (AM #103498). Use 5 µl of the diluted ChIP DNA per qPCR reaction.
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 6 below). If the qPCR enrichments are
satisfactory then the remaining 30 µl of ChIP DNA can be used for library generation.
Binding events detected / 1,000 cells

Poor Quality ChIP DNA
ess than -foldenrichment

Cell Line A
Cell Line B

ow signal
strengthigh background

Negative Control primer

Positive Control primer

Binding events detected / 1,000 cells

High Quality ChIP DNA
Cell Line A
Cell Line B

ow background and morethan -fold enrichment

Negative Control primer

Positive Control primer

Figure 6: Comparison of qPCR results showing good versus poor enrichment over negative control primers.
Data shows qPCR results analyzed using the ChIP-IT qPCR Analysis Kit with the Human Negative Control Primer as a reference and a gene-specific positive control primer. In the top image the negative control primer set
for Cell Line A gives high background levels with Binding events detected per 1,000 cells above a value of 2,
while Cell Line B has positive control signal levels below 5 Binding events detected per 1,000 cells. The bottom image shows low background in the negative control primer set with Binding events detected per 1,000
cells below a value of 2. The fold enrichment of the positive control primer set exceeds 5-fold. Only the ChIP
DNA from the bottom sample is recommended for use in ChIP-Seq.

D. Library Generation
ChIP DNA from the ChIP-IT High Sensitivity Kit has been extensively validated for ChIP-Seq using
the Illumina® platform. ChIP DNA can be used to generate single or paired-end libraries using
the standard Illumina® library protocols. Commercially available library preparation kits can beused to generate the NGS library. Select a library preparation kit compatible with the sequencing
platform to be used.
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23

Appendix
Section I: 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/)• Primers that dimerize should be avoided, as they will be bound by SYBR Green, which willcompromise accurate quantitation.
• 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 optimallyat 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 (from Section B Step 12 for cultured cell samples or Section D
Step 15 for tissue samples) 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, youwill need 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.

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24

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 (from Section B Step 12 for cultured cell samples or Section D
Step 15 for tissue samples) 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 (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 of theentire ChIP reaction by the volume used in the qPCR reaction (e.g. if you eluted ChIP
DNA in 200 µl and used 5 µl in the qPCR reaction the formula is 200/5 = 40). Then,
multiply the average qPCR quantity by this number (e.g. qPCR quantity in ng x 40).

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%.

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25Section J. 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. After formaldehyde fixation and centrifugation (intact cell pellet), -80°C.
2. After chromatin shearing, -80°C.
3. After DNA clean up, -20°C.

After sonication shearing and centrifugation,a viscous or cloudy
layer is visible in thechromatin.

Depending upon the cell type, lipid or glycogen layers may be seen after centrifugation. For example, fatty tissues may have a lipid layer. Avoid such layers
when you remove the supernatant. However, if the whole supernatant is cloudy,
it should not interfere with the IP reaction.

Poor yield of shearedchromatin.

Insufficient cell numbers were used. Repeat chromatin preparation using alarger number of cells.
Nuclei not released. It is highly recommended to perform dounce homogenization, even when using sonication. Use a dounce homogenizer with a small
clearance pestle (Active Motif Catalog Nos. 40401 & 40415). Monitor cell lysis
under a microscope. Generally, the more cells that are lysed, the higher the
sheared chromatin yield.
Sonication samples were emulsified. Avoid emulsification by turning up thepower of the sonicator gradually. If a chromatin preparation becomes emulsified inadvertently, discontinue shearing and centrifuge the sample for 4 minutes
at 8,000 rpm in a 4°C microcentrifuge to remove trapped air.
Use fresh formaldehyde when preparing Complete Cell Fixation Solution andComplete Tissue Fixation Solution.
Buffers were not scaled proportionally to the size of the sample. Use the chartin Cell Growth Recommendations to scale up or down chromatin preparation.

Shearing efficiency
is not clear from gelanalysis.

Material is stuck in the wells, and smears or streaks are seen from the top
to bottom of the lane. The sheared chromatin needs to have the cross-links
reversed, protein removed (Proteinase K) and RNA removed (RNase), followed
by DNA purification.
High molecular weight products. Decrease the size of the fragments by resonicating the sample. If an alternative reverse cross-linking method was used,or the 20 minute incubation at 100°C in NaCl was omitted prior to running the
agarose gel for analysis, please repeat the input chromatin preparation andfollow the manual instructions.

Performing ChIP witha large volume ofchromatin.

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 asingle large sample. Do not perform a single scaled-up reaction, as the capture
efficiency is lower.

ChIP DNA does not turnbright yellow following
the addition of 3 M
sodiium acetate

If the color is light orange or violet, this indicates the pH is too high. Add more
3 M sodium acetate 5 µl at a time, mixing after addition until the color is brightyellow. This step is crucial to the success of DNA binding and purification.
For a full color image please see the manual for Active Motif’s Chromatin IP
DNA Purification Kit Catalog No. 58002 available online at our website www.
activemotif.com.

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Problem/question

Recommendation

High background.

Chromatin not sheared enough. Shearing should produce DNA fragmentsthat are small enough to exclude background from neighboring chromosomal
sequences, but still large enough that there is a good possibility your ampliconremains intact. We recommend 200-1200 bp fragments. If the DNA fragments
are too large, the background is increased. Consider increasing the number ofpulses for sonication. Check the fragment size on a gel to assess your shearing
efficiency.
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 as increased background.

Poor or no enrichmentwith target antibody.

Too little chromatin. Generally, we recommend using 10 - 30 µg of chromatin per ChIP reaction. For highly abundant, DNA-associated targets such as
histones, chromatin from as little as 1000 cells or 6.7 ng can be used. For very
low abundance transcription factors, chromatin from as little as 50,000 cells or
333 ng can be used. ChIP reactions should not exceed 50 µg per IP reaction. 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, and that
equal mass quantities of chromatin are used in each ChIP.
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 is masked by other proteins in a larger complex. To assist in ChIP
validating an antibody, it is very useful to use a positive control antibody such
as Histone H3K4me3 (Catalog No. 39915) and a negative IgG from the same
species, and primers that have been proven to work in 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 variablebinding affinities to protein G, which are pH dependent; the optimal pH mayvary for each Ig, For those with low to medium affinity, capture efficiency byprotein 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 your IgG 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 is bound by the antibody target. Identify other binding sites.

No PCR products for the Increase the amount of chromatin used in the ChIP reaction, the amount of
ChIP’d samples (but the antibody used, or both.
Input DNA yields theUse a different antibody.
correct PCR product)
No PCR products withreal-time PCR

Confirm the species specificity and efficiency of your primers. You may need toredesign your primers. Primers that work in end point PCR do not always work
in qPCR.
No ethanol in DNA Purification Wash Buffer. Make sure that ethanol has been
added to the DNA Purification Wash Buffer prior to first use.

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Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
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

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com

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