CST Guide:

09
Section II: ANTIBODY APPLICATIONS
Chromatin Immunoprecipitation
(ChIP)
Optimal antibody concentration is critical for efficient ChIP.
Using either too much or too little antibody can result in significantly lower enrichment, potentially leading
to difficulty in detecting a low frequency, low stability binding event. Shown below are antibody titration
data generated as part of CST’s rigorous ChIP validation process for p53 (1C12) Mouse mAb #2524.
chapter 09: Chromatin Immunoprecipitation (ChIP)
The ChIP assay is a powerful and versatile technique used for probing protein-DNA interactions within
the natural chromatin context of the cell. The ChIP procedure isolates protein-DNA complexes that have
been chemically crosslinked and harvested, using an antibody to enrich for specific protein associations
by immunoprecipitation. After reversing the crosslinks, the DNA associated with a particular protein
can be analyzed by PCR or ChIP-Seq.
The success or failure of a ChIP experiment is highly dependent on the integrity of the chromatin, the
quality of the epitope, and the specificity of the antibody. This is especially true for low abundance,
low stability interactions—such as the binding of transcription factors (e.g., TCF4) and cofactors (e.g.,
Ezh2) that may fall under the detection threshold if the integrity of the protein or DNA is compromised,
or if the immunoenrichment relies on an antibody that is not highly specific to the target of interest.
ChIP Tips for Success
Enzymatic digestion better preserves chromatin integrity.
Unlike sonication, enzymatic digestion using micrococcal nuclease does not expose the sample to
harsh denaturing conditions (high heat and detergent), so it gently fragments the chromatin while
protecting the integrity of the protein-DNA complex. As shown below, enzyme-digested chromatin
displays a more robust enrichment of target DNA loci than does sonicated chromatin, particularly
with low frequency interactions.
Titration identifies the optimal amount of antibody to use per IP.
% of total input chromatin
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
1 µl
Optimal Antibody Concentration
2.5 µl
5 µl
10 µl
p53 (1C12) Mouse mAb #2524
20 µl
40 µl
Normal Rabbit
IgG #2729
SimpleChIP ® Enzymatic
Chromatin IP Kit (Magnetic
Beads) #9003: Chromatin IPs
were performed with cross-linked
chromatin from 4 x 10 6 HCT 116 cells
treated with UV (100 J/m 2 followed
by a 3 hr recovery) and the indicated
amounts of p53 (1C12) Mouse mAb
#2524 using #9003. The enriched
DNA was quantified by real-time
PCR using SimpleChIP ® Human
CDKN1A Promoter Primers #6449,
human MDM2 intron 2 primers,
and SimpleChIP ® Human α Satellite
Repeat Primers #4486. The amount
of immunoprecipitated DNA in each
sample is presented as a percent of
the total input chromatin.
CDKN1A
MDM2
Sat1α
The right positive control is essential for assay reliability.
Target DNA loci are
immunoprecipitated
better from enzymedigested
chromatin
than sonicated
chromatin.
SimpleChIP ® Plus Enzymatic
Chromatin IP Kit (Magnetic Beads)
#9005: Chromatin IPs were performed
with enzyme-digested or sonicated
chromatin and the indicated ChIPvalidated
antibodies using #9005.
The enriched DNA was quantified by
real-time PCR using primers to the
designated loci. The amount of immunoprecipitated
DNA in each sample
is presented as a percent of the total
input chromatin.
GAPDH RPL30 HoxA1 HoxA2
SimpleChIP
Sonicated
% of total input chromatin
% of total input chromatin
35
30
25
20
15
10
5
0
6
5
4
3
2
High Abundance,
High Stability Interactions
Histone H3 (D2B12) XP ® Rabbit
mAb (ChIP Formulated) #4620
Low Abundance,
Low Stability Interactions
Rpb1 CTD (4H8)
Mouse mAb #2629
Tri-Methyl-Histone H3 (Lys4)
(C42D8) Rabbit mAb #9751
Tri-Methyl-Histone H3 (Lys27)
(C36B11) Rabbit mAb #9733
A Histone H3 antibody will immunoprecipitate all genomic loci, whether transcriptionally active or
inactive, so it functions as a universal control for tracking assay efficiency and reagent performance.
Although Rpb1 antibody is often used as a positive control in other ChIP kits, it only binds active loci
and therefore may not be suitable when examining transcriptionally inactive regions of the genome.
H3 is a more reliable control than Rpb1.
% of total input chromatin
20
18
16
14
12
10
8
6
4
2
0
Histone H3 (D2B12) XP ® Rabbit
mAb (ChIP Formulated) #4620
Rpb1 CTD (4H8) Mouse mAb #2629 Normal Rabbit IgG #2729
Histone H3 (D2B12) XP ® Rabbit
mAb (ChIP Formulated) #4620:
ChIP was performed with 10 μg
of cross-linked chromatin and the
indicated antibodies. The enriched
DNA was quantified by qPCR. The
amount of immunoprecipitated DNA
in each sample is presented as a
percent of the total input chromatin.
γ-actin
GAPDH
RPL30
HoxA1
HoxA2
MYT1
1
0
Ezh2 (D2C9) XP ®
Rabbit mAb #5246
SUZ12 (D39F6) XP ®
Rabbit mAb #3737
Normal Rabbit IgG #2729
Benefits of Enzyme-based ChIP
To learn more about the major advantages of enzymatic digestion using micrococcal nuclease compared to sonication,
please go to www.cellsignal.com/chipbenefits
194 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstchip
195