ChIP protocol for cross-linking chromatin immunoprecipitation (X-ChIP)

Image 1 (above): 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.

Tip: Sonicating for too long will disrupt nucleosome-DNA interactions therefore the band size should not be smaller than 200 bp.

3. Calculate DNA concentration and measure DNA fragmentation size

The sonicated chromatin samples can be used to calculate the DNA concentration for subsequent IPs and measure DNA fragment size. 

3.1 Add 70 µL of elution buffer to the 50 µL of chromatin. 

3.2 Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.

Why use RNase A? 

Samples are treated with RNase A as high levels of RNA will interfere with DNA purification when using the PCR purification kit. Yields can be severely reduced as the columns become saturated.

3.3 Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.

Why use proteinase K?

Samples are treated with proteinase K, which cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. Cross-links between proteins and DNA are disrupted which aids DNA purification.

3.4 Purify DNA using a PCR purification kit or phenol: chloroform extraction.

3.5 To determine the DNA concentration, transfer 5 μL of the purified DNA into a tube containing 995 μL TE to give a 200-fold dilution and read the OD260. The concentration of DNA in μg/mL is OD260 x 10,000. This is used to calculate the DNA concentration of the chromatin preparation. Run purified DNA in a 1.5% agarose gel with a 100 bp DNA marker to determine fragment size.

4. Chromatin immunoprecipitation

4.1 Use the chromatin prepared from Step 2.2. Approximately 25 μg of DNA per IP is recommended. Dilute each sample 1:10 with RIPA Buffer. You will need one sample for the specific antibody and one sample for the control (beads only). Remove 50 µL of chromatin to serve as your input sample and store it at -20°C until further use.

4.2 Add the primary antibody to all samples, except the beads-only control, and rotate at 4°C for 1 h. The amount of antibody to be added should be determined empirically; 1 - 10 μg of antibody per 25 μg of DNA often works well.

4.3 Preparation of protein A/G beads: if using both Protein A and Protein G beads, mix an equal volume of Protein A and Protein G beads and wash three times in RIPA Buffer. Aspirate RIPA buffer and add single-stranded herring sperm DNA to a final concentration of 75 ng/μL beads and BSA to a final concentration of 0.1 μg/μL beads. Add RIPA buffer to twice the bead volume and incubate for 30 min with rotation at RT. Wash once with RIPA buffer and add RIPA buffer to twice the bead volume.

4.4 Add 60 µL of blocked protein A/G beads to all samples and IP overnight with rotation at 4°C.

Tip: Protein A beads, protein G beads, or a mix of both should be used. Table 1 shows the affinity of protein A and G beads to different immunoglobulin isotypes.

4.5 Centrifuge the immunoprecipitated samples for 1 min at 2,000 x g and remove the supernatant.

4.6 Perform the following washes: once in low salt wash buffer, once in high salt wash buffer, once in LiCl wash buffer. After each wash, centrifuge for 1 min at 2,000 x g and remove the supernatant.

Tip: If the high background is observed additional washes may be needed. Alternatively, the sonicated chromatin may be pre-cleared by incubating with the Protein A/G beads for 1 h prior to Step 4.2. Any non-specific binding to the beads will be removed during this additional step. Transfer the supernatant (sonicated chromatin) to a new tube and incubate with the antibody and beads as described in Step 4.2 onwards.

5. Elution and reversal of DNA cross-links

5.1 Elute DNA by adding 120 μL of elution buffer to the protein A/G beads and vortex slowly for 15 min at 30°C.

5.2 Centrifuge for 1 min at 2,000 x g and transfer the supernatant into a fresh tube.

5.3 Add 4.8 µL of 5 M NaCl and 2 µL RNase A (10 mg/mL) and incubate while shaking at 65°C overnight.

5.4 Add 2 µL proteinase K (20 mg/mL) and incubate while shaking at 60°C for 1 h.

5.5 The DNA can be purified using a PCR purification kit or phenol-chloroform extraction.

6. Analysis of DNA levels in ChIP-qPCR or ChIP-seq

6.1 DNA levels are often measured by real-time quantitative PCR. Primers and probes are often designed using software provided with the real-time PCR apparatus or using an online design tool. A selection of pre-designed primers and probes is available on our website.

6.2 Alternatively, analysis can be done by sequencing when using the ChIP-seq method. DNA produced using this protocol is suitable for use as an input for sequencing library prep.

Tip: We recommend using our troubleshooting tips to optimize this protocol for your particular experimental conditions.

Download or print the full protocol

Appendix: Solution buffer recipes

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  

Appendix: Affinity of Protein A and G beads to different immunoglobin isotypes across species