Protocol for Chromatin Immunoprecipitation (ChIP)
This is an abbreviated protocol to highlight the main points of ChIP using the ChromataChIP kit (NBP1-71709). A more detailed protocol can be found on our website www.novusbio.com or in online datasheets of ChromataChIP kits.
- Dilute each sheared chromatin sample 1:10 with IP dilution buffer containing protease inhibitors. Save an undiluted input sample at 4°C for the reverse crosslinking step.
- Add 2 µg of the antibody of interest into each of the diluted samples. It is also recommended to run the separate following controls:
Positive Control Antibody: An antibody known to work well in ChIP with your primer set.
Negative Control Antibody: A non-specific or isotype control antibody.
- Rotate the tubes overnight at 4°C.
- Add 25 µL of washed and suspended Protein A/G magnetic beads to each IP sample.
- Rotate the tube for 1 hour at 4°C.
- Pellet the magnetic beads with a magnetic separator and remove the supernatant. Add 500 μL of cold Wash buffer 1 and rotate for 5 minutes at 4°C. Pellet the magnetic beads with magnetic separator and discard supernatant.
- Add 500 μL cold Wash buffer 2 and rotate for 5 minutes at 4°C. Pellet the magnetic beads with magnetic separator and discard supernatant.
- Add 500 μL cold wash buffer 3 and rotate for 5 minutes at 4°C. Pellet the magnetic beads with magnetic separator and discard supernatant.
- Add 500 μL cold wash buffer 4 and rotate for 5 minutes at 4°C. Pellet the magnetic beads with magnetic separator and discard supernatant.
- Elute the antibody-protein complex by adding 200 μL of IP elution buffer and rotate at room temperature for 15 minutes. Pellet the magnetic beads with magnetic separator and keep the supernatant.
Reverse Cross-linking and DNA purification
- Add 8 μL of 5M NaCl to each sample. For the input control that did not go through the IP and wash steps, add 160 μL of IP Elution Buffer and 8 μL of 5M NaCl.
- Incubate the sample at 95°C for 15 minutes.
- . After the completion of Step 1, add 2 μL of Proteinase K to the samples and incubate at 62°C for 2 hours to overnight.
- Incubate at 95°C for 10 minutes to deactivate Proteinase K.
- The DNA samples can now be purified with spin columns or by phenol/chloroform extraction.
DNA PCR Amplification
- Purified DNA can now be measured by PCR. Quantitative real-time PCR is the preferable method of amplification due to its sensitivity. The method described below uses a 2X SYBR green reaction mix containing all necessary components (dNTPs, DNA polymerase, buffers). It is recommended to run each PCR reaction in triplicate for each sample. Samples to be assayed include: immunoprecipitated sample from the antibody of interest, the positive control sample the negative control sample, and the purified input control. Forward and reverse primers are also needed for each region of interest that will be amplified. Each sample will use 2 μl of purified DNA as template.
- For the input control fraction only, dilute the template to 1% of the original concentration (1:100 dilution). All other samples are left undiluted.
- It is best to first create a PCR master mix for each primer set and dispense the mix into each reaction well, then add the template last. In the case of your positive control primer set master mix, each reaction will contain the following:
7 μL of DNase free water
1 μL of 10 μM primers (final concentration 0.5 μM)
10 μL of 2x SYBR reaction mix
2 μL of purified DNA template
- Perform real time PCR according to manufacturer’s recommendations for the SYBR reaction mix.
Chromatin Immunoprecipitation (ChIP) Troubleshooting