Store at RT, 4C,

and 20C

SimpleChIP Plus Enzymatic

Chromatin IP Kit (Magnetic Beads)
31 Kit
n

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#9005

(30 Immunoprecipitations)

rev. 10/19/12

This product is intended for research purposes only. This product is not intended to be used for
therapeutic or diagnostic purposes in humans or animals.

Box 1 contains the following buffers/reagents:

Box 2 contains the following buffers/reagents:

Components to be stored at 4C:

Components to be stored at -20C:

Glycine Solution (10X)

100 ml

Protease Inhibitor Cocktail (200X)

2 X 750 l
50 l

indicated on the individual tube labels.

Note: Buffer A (4X), Buffer B (4X), and ChIP-Grade Protein G

Magnetic Beads contain 0.05% sodium azide.

Buffer A (4X)

25 ml

RNAse A (10 mg/ml)

Buffer B (4X)

25 ml

Micrococcal Nuclease (2000 gel units/l)

60 l

Reagents not Supplied:

ChIP Buffer (10X)

20 ml

Proteinase K (20 mg/ml)

100 l

Formaldehyde (37% Stock)

ChIP Elution Buffer (2X)

7 ml

SimpleChIP Human RPL30 Exon 3 Primers

150 l

Ethanol (96-100%)

5 M NaCl

3 ml

SimpleChIP Mouse RPL30 Intron 2 Primers

150 l

Isopropanol

0.5 M EDTA

1 ml

100 l

1X PBS

ChIP-Grade Protein G Magnetic Beads

1 ml

Histone H3 (D2B12) XP Rabbit mAb

(ChIP Formulated) #4620

Components to be stored at room temperature:

DNA Binding Reagent A (add 12 ml isopropanol
before use)

12 ml

Normal Rabbit IgG #2729 (1 g/l)

50 l

1M DTT

200 l

6 ml Specificity/Sensitivity: The SimpleChIP Plus Enzymatic

Chromatin IP Kit can be utilized with any ChIP-validated
DNA Elution Reagent C
2 X 1 ml antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells and tissue
DNA Spin Columns
36 samples (see Figures 1 and 2). The positive control Histone
H3 Antibody recognizes many different species of the highly
conserved Histone H3 protein, including human, mouse, rat
Description: The SimpleChIP Plus Kit provides enough
and monkey. Primer sets are included for the human and mouse
reagents to perform up to 30 immunoprecipitations and is
positive control RPL30 gene loci; however, the use of other
optimized for 25 mg of tissue or 4 X 106 cultured cells per
immunoprecipitation. Kit components are stable for 1 year from species with the kit requires the design of additional control
primer sets.
date of shipment when stored as directed.
DNA Wash Reagent B (add 24 ml ethanol before
use)

Histone H3 (D2B12) XP Rabbit mAb (ChIP Formulated) #4620

Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751
Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733
Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb #9649
Rpb1 CTD (4H8) Mouse mAb #2629
Normal Rabbit IgG #2729

Taq DNA polymerase

dNTP mix
6-Tube Magnetic Separation Rack #7017

Please visit www.cellsignal.com/technologies/

chip.html for a complete listing of recommended
companion products.

0.025

Signal relative to input

0.7
0.6
0.5
0.4
0.3
0.2

0.02
0.015
0.01
0.005

0.1
0

Nuclease-free water

Ezh2 (D2C9) XP Rabbit mAb #5246

SUZ12 (D39F6) XP Rabbit mAb #3737
RING1B (D22F2) XP Rabbit mAb #5694
PCAF (C14G9) Rabbit mAb #3378
Normal Rabbit IgG #2729

0.8

Signal relative to input

GAPDH

RPL30

HoxA1

HoxD10

GAPDH

RPL30

HoxA1

HoxD10

FIGURE 1. Mouse brain was cross-linked and disaggregated into a single-cell suspension using a Dounce homogenizer. The chromatin was prepared and digested, and chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP Mouse GAPDH Intron 2 Primers
#8986, SimpleChIP Mouse RPL30 Intron 2 Primers #7015, SimpleChIP Mouse HoxA1 Promoter Primers #7341, and SimpleChIP Mouse HoxD10 Exon 1 Primers #7429. The amount
of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

SimpleChIP kit co-developed by

CST and NEB; Antibodies provided
by CST; Enzymes provided by NEB.

page 1 of 8

2012 Cell Signaling Technology, Inc.

SimpleChIP, XP and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc.

Storage: Please store components at the temperatures

Histone H3 (D2B12) XP Rabbit mAb (ChIP Formulated) #4620

Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751
Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb #9733
Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mAb #9649
Rpb1 CTD (4H8) Mouse mAb #2629
Normal Rabbit IgG #2729
0.03

0.35

Signal relative to input

Signal relative to input

0.4

0.3
0.25
0.2
0.15
0.1

0.025
0.02
0.015
0.01
0.005

0.05
0

Ezh2 (D2C9) XP Rabbit mAb #5246

SUZ12 (D39F6) XP Rabbit mAb #3737
RING1B (D22F2) XP Rabbit mAb #5694
PCAF (C14G9) Rabbit mAb #3378
Normal Rabbit IgG #2729

GAPDH

AFM

HoxA1

HoxD10

GAPDH

AFM

HoxA1

HoxD10

FIGURE 2. Mouse liver was cross-linked and disaggregated into a single-cell suspension using a tissue disaggregator. The chromatin was prepared and digested, and chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP Mouse GAPDH Intron 2 Primers
#8986, SimpleChIP Mouse AFM Intron 2 Primers #7269, SimpleChIP Mouse HoxA1 Promoter Primers #7341, and SimpleChIP Mouse HoxD10 Exon 1 Primers #7429. The amount of
immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1).

1500
1200
1000
900
800
700
600
500
400
300
200
100
1

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page 2 of 8

2012 Cell Signaling Technology, Inc.

FIGURE 3. Mouse brain or mouse liver were cross-linked and

disaggregated into a single-cell suspension using a Dounce homogenizer or tissue disaggregator, respectively. The chromatin
was prepared and digested, and chromatin immunoprecipitations were performed using the indicated ChIP-validated
antibodies. DNA was purified and 10 l was separated by
electrophoresis on a 1% agarose gel and stained with ethidium
bromide. The majority of chromatin from both brain (lane 1)
and liver (lane 2) was digested to 1 to 5 nucleosomes in length
(150 to 900 bp).

Introduction:
The chromatin immunoprecipitation (ChIP) assay is a powerful
and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This
assay can be used to identify multiple proteins associated with
a specific region of the genome, or the opposite, to identify
the many regions of the genome associated with a particular
protein (3-6). In addition, the ChIP assay can be used to define
the spatial and temporal relationship of a particular proteinDNA interaction. For example, the ChIP assay can be used to
determine the specific order of recruitment of various protein
factors to a gene promoter or to measure the relative amount
of a particular histone modification across an entire gene locus
during gene activation (3,4). In addition to histone proteins, the
ChIP assay can also be used to analyze binding of transcription
factors, transcription co-factors, DNA replication factors, and
DNA repair proteins.
When performing the ChIP assay, cells or tissues are first fixed
with formaldehyde, a reversible protein-DNA cross-linking
agent that serves to fix or preserve the protein-DNA interactions occurring in the cell (see method overview) (1,2). Cells
are lysed and chromatin is harvested and fragmented using
either sonication or enzymatic digestion. The chromatin is then
subjected to immunoprecipitation using antibodies specific to a
particular protein or histone modification. Any DNA sequences
that are associated with the protein or histone modification of
interest will co-precipitate as part of the cross-linked chromatin
complex and the relative amount of that DNA sequence will be
enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the
DNA is purified. The enrichment of a particular DNA sequence
or sequences can then be detected by a number of different
methods.
Standard PCR methods are often employed to identify the
DNA sequences or regions of the genome associated with a
particular protein or histone modification (1,2). PCR is used to
measure the relative abundance of a particular DNA sequence
enriched by a protein-specific immunoprecipitation versus an
immunoprecipitation with a non-specific antibody control. PCR
products are run on an agarose or acrylamide gel to facilitate

quantification, and the level of enrichment of the DNA sequence

is determined relative to the total amount of input DNA (Percent
Input Method). The level of enrichment can also be expressed
as fold enrichment above background (enrichment relative
to that of the non-specific antibody control). Real-time PCR
provides a more accurate, gel-free system for the quantification of DNA enrichment. Alternatively, the ChIP assay can be
combined with genomic tiling micro-array (ChIP on chip)
techniques, sequencing, or cloning strategies, which allow for
genome-wide analysis of protein-DNA interactions and histone
modifications (5-8).
The SimpleChIP Plus Kit contains buffers and reagents
needed to perform the ChIP assay with mammalian cells and
tissue samples, and works for both histone modifications and
non-histone DNA-binding proteins. After cross-linking, tissues
are disaggregated into a single-cell suspension. Cells are then
lysed and the chromatin is fragmented by partial digestion
with Micrococcal Nuclease to obtain chromatin fragments of
1 to 5 nucleosomes in size. Enzymatic digestion of chromatin
is milder than sonication and eliminates problems due to
variability in sonication power and emulsification of chromatin
during sonication, which can result in incomplete fragmentation
of chromatin or loss of antibody epitopes due to protein denaturation and degradation. The chromatin immunoprecipitations
are performed using antibodies and either ChIP-Grade Protein
G Agarose Beads or ChIP-Grade Protein G Magnetic Beads.
After reversal of protein-DNA cross-links, the DNA is purified
using DNA purification spin columns provided in the kit. The
DNA purification spin columns combine the convenience of
spin-column technology with the selective binding properties
of a uniquely designed silica membrane that allows for efficient
recovery of DNA and removal of protein contaminants without
the need for phenol/chloroform extractions and ethanol precipitations. After DNA purification, the enrichment of particular
DNA sequences can be analyzed by a variety of methods, including standard PCR, quantitative real-time PCR, amplification
for ChIP on chip, sequencing, or cloning techniques.
In addition to providing buffers and reagents required to
perform the ChIP assay, the SimpleChIP Plus Kit provides
important controls that allow for user determination of a suc-

cessful ChIP experiment. The kit contains a positive control

histone H3 rabbit monoclonal antibody, a negative control
normal rabbit IgG antibody, and primer sets for PCR detection
of the ribosomal protein L30 (RPL30) gene locus (human and
mouse primer sets are included in the kit). Histone H3 is a
core component of chromatin in the cell and is bound to most
DNA sequences throughout the genome, including the RPL30
locus. Thus, immunoprecipitation of chromatin with the histone
H3 rabbit monoclonal antibody will enrich for the RPL30 gene,
while immunoprecipitation with the normal rabbit IgG antibody
will not result in RPL30 gene enrichment. This enrichment
can be quantified using either standard PCR or quantitative
real-time PCR methods with the RPL30 primer sets provided
in the kit. Importantly, since histone H3 is bound to most DNA
sequences throughout the genome, the Histone H3 Rabbit mAb
serves as a positive control IP for almost any locus studied,
giving the user even more confidence that their ChIP experiment was performed successfully.
The SimpleChIP Plus Kit provides enough reagents to perform
up to 30 immunoprecipitations and is optimized for 25 mg of
tissue or 4 X 106 cultured cells per immunoprecipitation. A
ChIP assay can be performed in as little as two days and can
easily be scaled up or down for use with more or less tissue
or cells.

Background References:
(1) Orlando, V. (2000) Trends Biochem Sci 25, 99104.
(2) K uo, M.H. and Allis, C.D. (1999) Methods 19, 42533.
(3) Agalioti, T. et al. (2000) Cell 103, 66778.
(4) Soutoglou, E. and Talianidis, I. (2002) Science 295,
19014.
(5) Mikkelsen, T.S. et al. (2007) Nature 448, 55360.
(6) Lee, T.I. et al. (2006) Cell 125, 30113.
(7) Weinmann, A.S. and Farnham, P.J. (2002) Methods
26, 3747.
(8) W
 ells, J. and Farnham, P.J. (2002) Methods 26, 4856.

Method Overview
Cells are fixed with formaldehyde to cross-link histone and
non-histone proteins to DNA.

Chromatin is digested with Micrococcal Nuclease into 150-900

bp DNA/protein fragments.

Antibodies specific to histone or non-histone proteins are added

and the complex co-precipitates and is captured by Protein G
Agarose or Protein G magnetic beads.

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page 3 of 8

2012 Cell Signaling Technology, Inc.

Cross-links are reversed, and DNA is purified and ready for

analysis.

#9005

Chromatin Immunoprecipitation Protocol

C Tissue Disaggregation Using a Dounce Homogenizer:

When harvesting tissue, remove unwanted material such as fat and necrotic material from
the sample. Tissue can then be processed and cross-linked immediately, or frozen on dry
ice for processing later. For optimal chromatin yield and ChIP results, use 25 mg of tissue
for each immunoprecipitation to be performed. The chromatin yield does vary between
tissue types and some tissues may require more than 25 mg for each immunoprecipitation.
Please see Appendix A for more information regarding the expected chromatin yield for different types of tissue. One additional chromatin sample should be processed for Analysis
of Chromatin Digestion and Concentration (Section IV).
Before starting:

R emove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine
Solution. Make sure PIC is completely thawed.
Prepare 3 ml of Phosphate Buffered Saline (PBS) + 15 l 200X PIC per 25 mg
of tissue to be processed and place on ice.
Prepare 45 l of 37% formaldehyde per 25 mg of tissue to be processed
and keep at room temperature. Use fresh formaldehyde that is not past the
manufacturers expiration date.

A Cross-linking
1. Weigh the fresh or frozen tissue sample. Use 25 mg of tissue for each IP to be
performed.
2. Place tissue sample in a 60 mm or 100 mm dish and finely mince using a clean
scalpel or razor blade. Keep dish on ice. It is important to keep the tissue cold to
avoid protein degradation.
3.

Transfer minced tissue to a 15 ml conical tube.

4.

Add 1 ml of PBS + PIC per 25 mg tissue to the conical tube.

5.

T o crosslink proteins to DNA, add 45 l of 37% formaldehyde per 1 ml of PBS + PIC

and rock at room temp for 20 min. Final formaldehyde concentration is 1.5%.

6.

S top cross-linking by adding 100 l of 10x glycine per 1 ml of PBS + PIC and mix for
5 min at room temperature.

7.

Centrifuge tissue at 1,500 rpm in a bench top centrifuge for 5 min at 4C.

8.

Remove supernatant and wash one time with 1 ml PBS + PIC per 25 mg tissue.

9.

Repeat centrifugation at 1,500 rpm in a bench top centrifuge for 5 min at 4C.

10. R emove supernatant and resuspend tissue in 1 ml PBS + PIC per 25 mg tissue and
store on ice. Disaggregate tissue into single-cell suspension using a Medimachine
(Part B) or Dounce homogenizer (Part C).

B Tissue Disaggregation Using Medimachine from

BD Biosciences (part #340587)
1. 
Cut off the end of a 1000 L pipette tip to enlarge the opening for transfer of tissue
chunks.
2. 
Transfer 1 ml of tissue resuspended in PBS + PIC into the top chamber of a 50 mm
medicone (part #340592).

2012 Cell Signaling Technology, Inc.

3.

Grind tissue for 2 min according to manufacturers instructions.

1. Transfer tissue resuspended in PBS + PIC to a Dounce homogenizer.

2. 
Disaggregate tissue pieces with 20-25 strokes. Check for single-cell suspension by
microscope (optional).
3. 
Transfer cell suspension to a 15 ml conical tube and centrifuge at 1,500 rpm in a
bench top centrifuge for 5 min at 4C.
4. 
Remove supernatant from cells and immediately continue with Nuclei Preparation and
Chromatin Digestion (Section III).

II. Cell Culture Cross-linking and Sample Preparation:

For optimal ChIP results, use approximately 4 X 106 cells for each immunoprecipitation to
be performed. For HeLa cells, this is equivalent to half of a 15 cm culture dish containing
cells that are 90% confluent in 20 ml of growth medium. One additional sample should
be processed for Analysis of Chromatin Digestion and Concentration (Section IV). Include
one extra dish of cells in experiment to be used for determination of cell number using a
hemocytometer.
Before starting:



R emove and warm 200X Protease Inhibitor Cocktail (PIC) and 10X Glycine
Solution. Make sure PIC is completely thawed.
Prepare 2 ml of Phosphate Buffered Saline (PBS) + 10 l 200X PIC per 15 cm
dish to be processed and place on ice.
Prepare 40 ml of PBS per 15 cm dish to be processed and place on ice.
Prepare 540 l of 37% formaldehyde per 15 cm dish of cells to be processed
and keep at room temperature. Use fresh formaldehyde that is not past the
manufacturers expiration date.

1. To crosslink proteins to DNA, add 540 l of 37% formaldehyde to each 15 cm culture
dish containing 20 ml medium. Swirl briefly to mix and incubate 10 min at room
temperature. Final formaldehyde concentration is 1%. Addition of formaldehyde may
result in a color change of the medium.
2. Add 2 ml of 10X glycine to each 15 cm dish containing 20 ml medium, swirl briefly
to mix, and incubate 5 min at room temperature. Addition of glycine may result in a
color change of the medium.
3. For suspension cells, transfer cells to a 50 ml conical tube, centrifuge at 1,500 rpm
in a bench top centrifuge 5 min at 4C and wash pellet two times with 20 ml ice-cold
PBS. Remove supernatant and immediately continue with Nuclei Preparation and
Chromatin Digestion (Section III).
4.

F or adherent cells, remove media and wash cells two times with 20 ml ice-cold 1X
PBS, completely removing wash from culture dish each time.

5. A dd 2 ml ice-cold PBS + PIC to each 15 cm dish. Scrape cells into cold buffer.
Combine cells from all culture dishes into one 15 ml conical tube.
6. Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4C. Remove
supernatant and immediately continue with Nuclei Preparation and Chromatin Digestion (Section III).

III. Nuclei Preparation and Chromatin Digestion:

4. 
Collect cell suspension from the bottom chamber of the medicone using a 1 ml
syringe and 18 gauge blunt needle. Transfer cell suspension to a 15 ml conical tube
and place on ice.

One immunoprecipitation preparation (IP prep) is defined as 25 mg of disaggregated tissue

or 4 x 106 tissue culture cells.

5.

Before starting:

Repeat steps 2 to 4 until all the tissue is processed into a homogenous suspension.

6. 
If more grinding is necessary, add more PBS + PIC to tissue. Repeat steps 2 to 5 until
all tissue is ground into a homogeneous suspension.

7.

Check for single-cell suspension by microscope (optional).

8.

Centrifuge cells at 1,500 rpm in a bench top centrifuge for 5 min at 4C.

9. 
Remove supernatant from cells and immediately continue with Nuclei Preparation and
Chromatin Digestion (Section III).
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R emove and warm 200X Protease Inhibitor Cocktail (PIC) and 1 M DTT. Make
sure both are completely thawed and DTT crystals are completely in solution.
Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
Prepare 1 ml 1X Buffer A (250 l 4X Buffer A + 750 l water) + 0.5 l 1M DTT
+ 5 l 200X PIC per IP prep and place on ice.
Prepare 1.1 ml 1X Buffer B (275 l 4X Buffer B + 825 l water) + 0.55 l 1M
DTT per IP prep and place on ice.

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page 4 of 8

I. Tissue Cross-linking and Sample Preparation:

P repare 100 l 1X ChIP Buffer (10 l 10X ChIP Buffer + 90 l water) + 0.5 l
200X PIC per IP prep and place on ice.

1. R esuspend cells in 1 ml ice-cold Buffer A + DTT + PIC per IP prep. Incubate on ice
for 10 min. Mix by inverting tube every 3 min.
2. Pellet nuclei by centrifugation at 3,000 rpm in a bench top centrifuge for 5 min at 4C.
Remove supernatant and resuspend pellet in 1 ml ice-cold Buffer B + DTT per IP prep.
Repeat centrifugation, remove supernatant, and resuspend pellet in 100 l Buffer B +
DTT per IP prep. Transfer sample to a 1.5 ml microcentrifuge tube, up to 1 ml total per
tube.

V. Chromatin Immunoprecipitation:
For optimal ChIP results, use approximately 5 to 10 g of digested, cross-linked chromatin
(as determined in Section IV) per immunoprecipitation. This should be roughly equivalent
to a single 100 l IP prep from 25 mg of disaggregated tissue or 4 x 106 tissue culture
cells. Typically, 100 l of digested chromatin is diluted into 400 l 1X ChIP Buffer prior
to the addition of antibodies. However, if more than 100 l of chromatin is required per
IP, the cross-linked chromatin preparation does not need to be diluted as described below.
Antibodies can be added directly to the undiluted chromatin preparation for immunoprecipitation of chromatin complexes.

3. Add 0.5 l of Micrococcal Nuclease per IP prep, mix by inverting tube several times
and incubate for 20 min at 37C with frequent mixing to digest DNA to length of approximately 150-900 bp. Mix by inversion every 3 to 5 min. The amount of Micrococcal Nuclease required to digest DNA to the optimal length may need to be determined
empirically for individual tissues and cell lines (see Appendix B). HeLa nuclei
digested with 0.5 l Micrococcal Nuclease per 4 x 106 cells and mouse liver tissue
digested with 0.5 l Micrococcal Nuclease per 25 mg of tissue gave the appropriate
length DNA fragments (see Figure 3).

Before starting:

4. Stop digest by adding 10 l of 0.5 M EDTA per IP prep and placing tube on ice.

5. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4C

and remove supernatant.
6. R esuspend nuclear pellet in 100 l of 1X ChIP buffer + PIC per IP prep and incubate
on ice for 10 min.
7. Sonicate up to 500 l of lysate per 1.5 ml microcentrifuge tube with several pulses
to break nuclear membrane. Incubate samples for 30 sec on wet ice between pulses.
Optimal conditions required for complete lysis of nuclei can be determined by observing nuclei under light microscope before and after sonication. HeLa nuclei were
completely lysed after 3 sets of 20-sec pulses using a VirTis Virsonic 100 Ultrasonic
Homogenizer/Sonicator at setting 6 with a 1/8-inch probe. Alternatively, nuclei can be
lysed by homogenizing the lysate 20 times in a Dounce homogenizer; however, lysis
may not be as complete.
8. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4C.
9. Transfer supernatant to a new tube. This is the cross-linked chromatin preparation,
which should be stored at -80C until further use. Remove 50 l of the chromatin
preparation for Analysis of Chromatin Digestion and Concentration (Section IV).

IV. Analysis of Chromatin Digestion and Concentration

(Recommended Step)
1. To the 50 l chromatin sample (from Step 9 in Section III), add 100 l nuclease-free
water, 6 l 5 M NaCl ,and 2 l RNAse A. Vortex to mix and incubate samples at 37C
for 30 min.
2. To each RNAse A-digested sample, add 2 l Proteinase K. Vortex to mix and incubate
samples at 65C for 2 h.
3. Purify DNA from samples using DNA purification spin columns as described in Section VII.

2012 Cell Signaling Technology, Inc.

4. After purification of DNA, remove a 10 l sample and determine DNA fragment size
by electrophoresis on a 1% agarose gel with a 100 bp DNA marker. DNA should be
digested to a length of approximately 150-900 bp (1 to 5 nucleosomes; see Figure 3).
5. To determine DNA concentration, transfer 2 l of purified DNA to 98 l nuclease-free
water to give a 50-fold dilution and read the OD260. The concentration of DNA in g/ml is
OD260 x 2,500. DNA concentration should ideally be between 50 and 200 g/ml.
NOTE: For optimal ChIP results, it is highly critical that the chromatin is of appropriate size and concentration. Over-digestion of chromatin may diminish signal in the PCR
quantification. Under-digestion of chromatin may lead to increased background signal and
lower resolution. Adding too little chromatin to the IP may result in diminished signal in
the PCR quantification. A protocol for optimization of chromatin digestion can be found in
Appendix B.

1. In one tube, prepare enough 1X ChIP Buffer for the dilution of digested chromatin
into the desired number of immunoprecipitations: 400 l of 1X ChIP Buffer (40 l
of 10X ChIP Buffer + 360 l water) + 2 l 200X PIC per immunoprecipitation. When
determining the number of immunoprecipitations, remember to include the positive
control Histone H3 (D2B12) XP Rabbit mAb and negative control Normal Rabbit IgG
antibody samples. Place mix on ice.
2. To the prepared 1X ChIP buffer, add the equivalent of 100 l (5 to 10 g of chromatin)
of the digested, cross-linked chromatin preparation (from Step 9 in Section III) per
immunoprecipitation. For example, for 10 immunoprecipitations, prepare a tube
containing 4 ml 1X ChIP Buffer (400 l 10X ChIP Buffer + 3.6 ml water) + 20 l 200X
PIC + 1 ml digested chromatin preparation.
3. Remove a 10 l sample of the diluted chromatin and transfer to a microfuge tube. This
is your 2% Input Sample, which can be stored at -20C until further use (Step 1 in
Section VI).
4. For each immunoprecipitation, transfer 500 l of the diluted chromatin to a 1.5 ml microcentrifuge tube and add the immunoprecipitating antibody. The amount of antibody
required per IP varies and should be determined by the user. For the positive control
Histone H3 (D2B12) XP Rabbit mAb, add 10 l to the IP sample. For the negative
control Normal Rabbit IgG, add 1 l (1 g) to 2 l (2 g) to the IP sample. Incubate IP
samples 4 h to overnight at 4C with rotation.
5. 
Resuspend ChIP-Grade Protein G Magnetic Beads by gently vortexing. Immediately
add 30 l of Protein G Magnetic Beads to each IP reaction and incubate for 2 h at 4C
with rotation.
6. 
Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in
a Magnetic Separation Rack. Wait 1 to 2 min for solution to clear and then carefully
remove supernatant.
7. 
Wash Protein G Magnetic Beads by adding 1 ml of low salt wash to the beads and
incubate at 4C for 5 min with rotation. Repeat steps 6 and 7 two additional times for
a total of 3 low salt washes.
8. Add 1 ml of high salt wash to the beads and incubate at 4C for 5 min with rotation.
9. 
Pellet Protein G Magnetic Beads in each immunoprecipitation by placing the tubes in
a Magnetic Separation Rack. Wait 1 to 2 min for solution to clear and then carefully
remove supernatant. Immediately proceed to Section VI.

VI. Elution of Chromatin from Antibody/Protein G Magnetic

Beads and Reversal of Cross-links:
Before starting:

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R emove and warm 200X Protease Inhibitor Cocktail (PIC). Make sure PIC is
completely thawed.
Remove and warm 10X ChIP Buffer and ensure SDS is completely in solution.
Thaw digested chromatin preparation (from Step 9 in Section III) and place on
ice.
Prepare low salt wash: 3 ml 1X ChIP Buffer (300 l 10X ChIP Buffer + 2.7 ml
water) per immunoprecipitation. Store at room temperature until use.
Prepare high salt wash: 1 ml 1X ChIP Buffer (100 l 10X ChIP Buffer + 900 l
water) + 70 l 5M NaCl per immunoprecipitation. Store at room temperature
until use.

R emove and warm 2X ChIP Elution Buffer in a 37C water bath and ensure
SDS is in solution.

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page 5 of 8

#9005

Chromatin Immunoprecipitation Protocol (cont.)

#9005

Chromatin Immunoprecipitation Protocol (cont.)

Set a water bath or thermomixer to 65C.

Prepare 150 l 1X ChIP Elution Buffer (75 l 2X ChIP Elution Buffer + 75 l
water) for each immunoprecipitation and the 2% input sample.

1. Add 150 l of the 1X ChIP Elution Buffer to the 2% input sample tube and set aside at
room temperature until Step 6.
2. Add 150 l 1X ChIP Elution Buffer to each IP sample.
3. 
Elute chromatin from the antibody/Protein G Magnetic Beads for 30 min at 65C with
gentle vortexing (1,200 rpm). A thermomixer works best for this step. Alternatively,
elutions can be performed at room temperature with rotation, but may not be as
complete.
4. 
Pellet Protein G Magnetic Beads by placing the tubes in a Magnetic Separation Rack
and wait 1 to 2 min for solution to clear.
5. Carefully transfer eluted chromatin supernatant to a new tube.
6. To all tubes, including the 2% input sample from Step 1, reverse cross-links by adding 6 l 5M NaCl and 2 l Proteinase K, and incubate 2 h at 65C. This incubation
can be extended overnight.
7. Immediately proceed to Section VII. Alternatively, samples can be stored at -20C.
However, to avoid formation of a precipitate, be sure to warm samples to room
temperature before adding DNA Binding Reagent A (Section VII, Step 1).

VII. DNA Purification Using Spin Columns:

Before starting:

A dd 12 ml of isopropanol to DNA Binding Reagent A and 24 ml of ethanol

(96-100%) to DNA Wash Reagent B before use. These steps only have to be
performed once prior to the first set of DNA purifications.
R emove one DNA purification spin column and collection tube for each DNA
sample from Section VI.

1. Add 600 l DNA Binding Reagent A to each DNA sample and vortex briefly.

4 volumes of DNA Binding Reagent A should be used for every 1 volume of sample.

2. Transfer 375 l of each sample from Step 1 to a DNA purification spin column in
collection tube.

A Hot-Start Taq polymerase is recommended to minimize the risk of nonspecific PCR products.

P CR primer selection is critical. Primers should be designed with close adherence to the following criteria:

Primer length:

24 nucleotides

Optimum Tm:

60C

Optimum GC:

50%

Amplicon size:

150 to 200 bp (for standard PCR)

80 to 160 bp (for real-time quantitative PCR)

Standard PCR Method:

1. Label the appropriate number of 0.2 ml PCR tubes for the number of samples to be
analyzed. These should include the 2% input sample, the positive control histone H3
sample, the negative control normal rabbit IgG sample, and a tube with no DNA to
control for DNA contamination.
2. Add 2 l of the appropriate DNA sample to each tube.
3. Prepare a master reaction mix as described below, making sure to add enough
reagent for two extra tubes to account for loss of volume. Add 18 l of master mix to
each reaction tube.
Reagent

Volume for 1 PCR Reaction (18 l)

Nuclease-free H2O

12.5 l

10X PCR Buffer

2.0 l

4 mM dNTP Mix

1.0 l

5 M RPL30 Primers

2.0 l

Taq DNA Polymerase

0.5 l

4. Start the following PCR reaction program:

a.

Initial Denaturation

95C

5 min

3. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.

b.

Denature

95C

30 sec

4. Remove the spin column from the collection tube and discard the liquid. Replace spin
column in the collection tube.

c.

Anneal

62C

30 sec

d.

Extension

72C

30 sec

e.

Repeat Steps b-d for a total of 34 cycles.

f.

Final Extension

72C

5 min

5. Transfer the remaining 375 l of each sample from Step 1 to the spin column in collection tube. Repeat Steps 3 and 4.
6. Add 700 l of DNA Wash Reagent B to the spin column in collection tube.
7. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.
8. Remove the spin column from the collection tube and discard the liquid. Replace spin
column in the collection tube.
9. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec.

5. Remove 10 l of each PCR product for analysis by 2% agarose gel or 10% polyacrylamide gel electrophoresis with a 100 bp DNA marker. The expected size of the
PCR product is 161 bp for human RPL30 and 159 bp for mouse RPL30.

10. Discard collection tube and liquid. Retain spin column.

Real-Time Quantitative PCR Method:

11. Add 50 l of DNA Elution Reagent C to each spin column and place into a clean
1.5 ml microcentrifuge tube.
13. Remove and discard DNA purification spin column. Eluate is now purified DNA.
Samples can be stored at -20C.

1. Label the appropriate number of PCR tubes or PCR plates compatible with the model
of PCR machine to be used. PCR reactions should include the positive control histone
H3 sample, the negative control normal rabbit IgG sample, a tube with no DNA to
control for contamination, and a serial dilution of the 2% input chromatin DNA
(undiluted, 1:5, 1:25, 1:125) to create a standard curve and determine the efficiency of
amplification.

VIII. Quantification of DNA by PCR:

2. Add 2 l of the appropriate DNA sample to each tube or well of the PCR plate.
3. Prepare a master reaction mix as described below. Add enough reagents for two extra
reactions to account for loss of volume. Add 18 l of reaction mix to each PCR reaction tube or well.

Recommendations:

Use Filter-tip pipette tips to minimize risk of contamination.

T he control primers included in the kit are specific for the human or mouse
RPL30 gene and can be used for either standard PCR or quantitative real-time
PCR. If the user is performing ChIPs from another species, it is recommended
that the user design the appropriate specific primers to DNA and determine the
optimal PCR conditions.
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12. Centrifuge at 14,000 rpm in a microcentrifuge for 30 sec to elute DNA.

#9005

Chromatin Immunoprecipitation Protocol (cont.)

Reagent

Volume for 1 PCR Reaction (18 l)

Nuclease-free H2O

6 l

5 M RPL30 Primers

2 l

2X SYBR-Green Reaction Mix

10 l

5. 
Analyze quantitative PCR results using the software provided with the real-time PCR
machine. Alternatively, one can calculate the IP efficiency manually using the Percent
Input Method and the equation shown below. With this method, signals obtained
from each immunoprecipitation are expressed as a percent of the total input chromatin.

4. Start the following PCR reaction program:

a.

Initial Denaturation

95C 3 min

b.

Denature

95C 15 sec

c.

Anneal and Extension:

60C 60 sec

d.

Repeat steps b and c for a total of 40 cycles.

Percent Input = 2% x 2(C[T] 2%Input Sample C[T] IP Sample)

C[T] = CT = Threshold cycle of PCR reaction

APPENDIX A: Expected Chromatin Yield

When harvesting cross-linked chromatin from tissue samples, the yield of chromatin
can vary significantly between tissue types. The table to the right provides a range for the
expected yield of chromatin from 25 mg of tissue compared to 4 x 106 HeLa cells, and the
expected DNA concentration, as determined in Section IV of the protocol. For each tissue
type, disaggregation using a Medimachine (BD Biosciences) or a Dounce homogenizer
yielded similar amounts of chromatin. However, chromatin processed from tissues
disaggregated using the Medimachine typically gave higher IP efficiencies than chromatin
processed from tissues disaggregated using a Dounce homogenizer. A Dounce homogenizer is strongly recommended for disaggregation of brain tissue, as the Medimachine
does not adequately disaggregate brain tissue into a single-cell suspension. For optimal
ChIP results, we recommend using 5 to 10 g of digested, cross-linked chromatin per
immunoprecipitation; therefore, some tissues may require harvesting more than 25 mg per
each immunoprecipitation.

Tissue/Cell

Total Chromatin Yield

Expected DNA Concentration

Spleen

20-30 g per 25 mg tissue

200-300 g/ml

Liver

10-15 g per 25 mg tissue

100-150 g/ml

Kidney

8-10 g per 25 mg tissue

80-100 g/ml

Brain

2-5 g per 25 mg tissue

20-50 g/ml

Heart

2-5 g per 25 mg tissue

20-50 g/ml

HeLa

10-15 g per 4 x 106 cells

100-150 g/ml

APPENDIX B: Optimization of Chromatin Digestion

1. Prepare cross-linked nuclei from 125 mg of tissue or 2 X 107 cells (equivalent of 5

IP preps), as described in Sections I, II, and III. Stop after Step 2 of Section III and
proceed as described below.

9. Clarify lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10 min at 4C.

10. Transfer 50 l of each of the sonicated lysates to new microfuge tubes.
11. To each 50 l sample, add 100 l nuclease-free water, 6 l 5 M NaCl and 2 l RNAse
A. Vortex to mix and incubate samples at 37C for 30 min.

2. Transfer 100 l of the nuclei preparation into 5 individual 1.5 ml microcentrifuge

tubes and place on ice.

12. To each RNAse A-digested sample, add 2 l Proteinase K. Vortex to mix and incubate
sample at 65C for 2 h.

3. A dd 3 l Micrococcal Nuclease stock to 27 l of 1X Buffer B + DTT (1:10 dilution of

enzyme).

13. Remove 20 l of each sample and determine DNA fragment size by electrophoresis on
a 1% agarose gel with a 100 bp DNA marker.

4. To each of the 5 tubes in Step 2, add 0 l, 2.5 l, 5 l, 7.5 l, or 10 l of the diluted
Micrococcal Nuclease, mix by inverting tube several times and incubate for 20 min at
37C with frequent mixing.

14. Observe which of the digestion conditions produces DNA in the desired range of
150-900 base pairs (1 to 5 nucleosomes, see Figure 3). The volume of diluted
Micrococcal Nuclease that produces the desired size of DNA fragments using this
optimization protocol is equivalent to 10 times the volume of Micrococcal Nuclease
stock that should be added to one immunoprecipitation preparation (25 mg of disaggregated tissue cells or 4 X 106 tissue culture cells) to produce the desired size of
DNA fragments. For example, if 5 l of diluted Micrococcal Nuclease produces DNA
fragments of 150-900 base pairs in this protocol, then 0.5 l of stock Micrococcal
Nuclease should be added to one immunoprecipitation preparation during the digestion of chromatin in Section III.

5. Stop each digest by adding 10 l of 0.5 M EDTA and placing tubes on ice.
2012 Cell Signaling Technology, Inc.

probe. Alternatively, nuclei can be lysed by homogenizing the lysate 20 times in a

Dounce homogenizer; however, lysis may not be as complete.

6. Pellet nuclei by centrifugation at 13,000 rpm in a microcentrifuge for 1 min at 4C

and remove supernatant.
7. R esuspend nuclear pellet in 200 l of 1X ChIP buffer + PIC. Incubate on ice for 10
min.
8. Sonicate lysate with several pulses to break nuclear membrane. Incubate samples
30 sec on wet ice between pulses. Optimal conditions required for complete lysis of
nuclei can be determined by observing nuclei under light microscope before and after
sonication. HeLa nuclei were completely lysed after 3 sets of 20-sec pulses using a
VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator set at setting 6 with a 1/8-inch
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15. If results indicate that DNA is not in the desired size range, then repeat optimization
protocol, adjusting the amount of Micrococcal Nuclease in each digest accordingly.
Alternatively, the digestion time can be changed to increase or decrease the extent of
DNA fragmentation.

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Optimal conditions for the digestion of cross-linked chromatin DNA to 150-900 base pairs
in length is highly dependent on the ratio of Micrococcal Nuclease to the amount of tissue
or number of cells used in the digest. Below is a protocol for determination of the optimal
digestion conditions for a specific tissue or cell type.

#9005

APPENDIX C: Troubleshooting Guide

Problem

Possible Causes

Recommendation

1. Concentration of the digested chromatin is too low (low chromatin yield).

Not enough tissue or cells were added to the chromatin digestion or

cell nuclei were not completely lysed after digestion.

Add additional chromatin to each IP to give at least 5 g/IP and

continue with protocol.
Weigh tissue or count a separate plate of cells prior to cross-linking
to determine accurate cell number. Some tissues may require
processing of more than 25 mg per IP. The amount of tissue can be
increased to 50 mg per IP, while still maintaining efficient chromatin
fragmentation and extraction
Increase the number of sonications following chromatin digestion.
Visualize cell nuclei under microscope before and after sonication to
confirm complete lysis of nuclei.

Tissue or cells may have been over cross-linked. Cross-linking for

longer than 10 min may inhibit digestion of chromatin.

Perform a time course at a fixed formaldehyde concentration. Shorten

the time of cross-linking to 10 min or less.

3. Chromatin is over-digested and

fragments are too small (exclusively
150 bp mono-nucleosome length).
Complete digestion of chromatin to
mono-nucleosome length DNA may
diminish signal during PCR quantification, especially for amplicons greater
than 150 bp in length.

Not enough cells or too much Micrococcal Nuclease added to the

chromatin digestion.

Weigh tissue or count a separate plate of cells prior to cross-linking

to determine accurate cell number. Add more tissue or cells, or less
Micrococcal nuclease to the chromatin digest. See Appendix B for
optimization of chromatin digestion.

4. No product or very little product in

the input PCR reactions.

Not enough DNA added to the PCR reaction or conditions are not
optimal.

Add more DNA to the PCR reaction or increase the number of

amplification cycles.

PCR amplified region may span nucleosome-free region.

Optimize the PCR conditions for experimental primer set using

purified DNA from cross-linked and digested chromatin. Design a
different primer set and decrease length of amplicon to less than
150 bp (see primer design recommendations in Section VIII).

Not enough chromatin added to the IP or chromatin is over-digested.

For optimal ChIP results, add 5 to 10 g chromatin per IP. See

recommendations for problems 1 and 3 above.

Not enough chromatin or antibody added to the IP reaction or IP

incubation time is too short.

Be sure to add 5 to 10 g of chromatin and 10 l of antibody to each

IP reaction and incubate with antibody over-night and an additional
2 h after adding Protein G beads.

Incomplete elution of chromatin from Protein G beads.

Elution of chromatin from Protein G beads is optimal at 65C with

frequent mixing to keep beads suspended in solution.

Too much or not enough chromatin added to the IP reaction. Alternatively, too much antibody added to the IP reaction.

For optimal ChIP results, add 5 to 10 g of chromatin and 10 l

of histone H3 antibody to each IP reaction. Reduce the amount of
normal rabbit IgG to 1 l per IP.

Too much DNA added to the PCR reaction or too many cycles of
amplification.

Add less DNA to the PCR reaction or decrease the number of PCR
cycles. It is very important that the PCR products are analyzed within
the linear amplification phase of PCR. Otherwise, the differences in
quantities of starting DNA cannot be accurately measured. Alternatively, quantify immunoprecipitations using real-time quantitative
PCR.

Not enough DNA added to the PCR reaction.

Add more DNA to the PCR reaction or increase the number of

amplification cycles.

Not enough antibody added to the IP reaction.

Typically a range of 1 to 5 g of antibody are added to the IP reaction; however, the exact amount depends greatly on the individual
antibody. Increase the amount of antibody added to the IP.

Antibody does not work for ChIP.

Find an alternate antibody source.

5. No product in the positive control

histone H3-IP RPL30 PCR reaction.

6. Quantity of product in the negative

control Rabbit IgG-IP and positive
control histone H3-IP PCR reactions is
equivalent (high background signal).

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Too many cells or not enough Micrococcal Nuclease was added to the Weigh tissue or count a separate plate of cells prior to cross-linking
chromatin digestion.
to determine accurate cell number. Add less tissue or cells, or more
Micrococcal nuclease to the chromatin digest. See Appendix B for
optimization of chromatin digestion.

7. No product in the Experimental

Antibody-IP PCR reaction.

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2. Chromatin is under-digested and

fragments are too large (greater than
900 bp). Large chromatin fragments
can lead to increased background and
lower resolution.