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Detailed procedure and tips for cross-linking ChIP with ChIP-seq and ChIP-qPCR methods.
View the X-ChIP protocol diagram.
ChIP-seq and ChIP-qPCR are powerful tools that allow the specific matching of proteins or histone modifications to regions of the genome. Chromatin is isolated and antibodies to the antigen of interest are used to determine whether the target binds to a specific DNA sequence or to map the distribution across the genome (microarray or DNA sequencing). This can be performed both spatially and temporally. This protocol provides specific details of how ChIP can be performed on cells.
The output DNA produced using this protocol can either be analyzed using qPCR in ChIP-qPCR or with sequencing in ChIP-seq.
More resources and products for ChIP
After reading this ChIP protocol, review more ChIP resources and products, or go straight to getting great ChIP data with:
- carefully validated ChIP antibodies, including recombinant rabbit monoclonals designed to give exactly the same performance year-after-year
- flexible Chromatin Extraction Kit ab117152 which is used to extract native, cross-linked, sheared or unsheared chromatin
- easy-to-use ChIP Kit ab500, or another kit from the ChIP kit product range.
To help you master ChIP assay, we've developed a free online ChIP training program.
We recommend using our troubleshooting tips to optimize this protocol for your particular experimental conditions.
Figure 1. U2OS cells were sonicated for 5, 10, 15 and 20 min. The fragment size decreases during the time course. The optimal fragment size is observed at 15 min. NOTE; sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.
Species Immunoglobulin isotype | Protein A | Protein G |
Human IgG1 | +++ | +++ |
IgG2 | +++ | +++ |
IgG3 | - | +++ |
IgG4 | +++ | +++ |
IgM | Use anti Human IgM | Use anti Human IgM |
IgE | - | + |
IgA | - | + |
Mouse IgG1 | + | +++ |
IgG2a | +++ | +++ |
IgG2b | ++ | ++ |
IgG3 | + | + |
IgM | Use anti human IgM | Use anti human IgM |
Rat IgG1 | - | + |
IgG2a | - | +++ |
IgG2b | - | ++ |
IgG2c | + | ++ |
Chicken all isotypes | - | ++ |
Cow all isotypes | ++ | +++ |
Goat all isotypes | - | ++ |
Guinea Pig all isotypes | +++ | ++ |
Hamster all isotypes | + | ++ |
Horse all isotypes | ++ | +++ |
Pig all isotypes | + | ++ |
Rabbit all isotypes | +++ | ++ |
Sheep all isotypes | - | ++ |
Table 1. The affinity of Protein A and G beads to different immunoglobin isotypes.
ChIP Lysis Buffer
50 mM HEPES-KOH pH7.5
140 mM NaCl
1 mM EDTA pH8
1% Triton X-100
0.1% Sodium Deoxycholate
0.1% SDS
Protease Inhibitors (add fresh each time)
RIPA Buffer
50 mM Tris-HCl pH8
150 mM NaCl
2 mM EDTA pH8
1% NP-40
0.5% Sodium Deoxycholate
0.1% SDS
Protease Inhibitors (add fresh each time)
Low Salt Wash Buffer
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM Tris-HCl pH 8.0
150 mM NaCl
High Salt Wash Buffer
0.1% SDS
1% Triton X-100
2 mM EDTA
20 mM Tris-HCl pH 8.0
500 mM NaCl
LiCl Wash Buffer
0.25 M LiCl
1% NP-40
1% Sodium Deoxycholate
1 mM EDTA
10 mM Tris-HCl pH 8.0
TE Buffer
10 mM Tris pH 8.0
1 mM EDTA
Elution Buffer
1% SDS
100mM NaHCO3