Chromatin Immunoprecipitation Protocol

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.

 

Chromatin Immunoprecipitation

  1. 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.
  2. 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.

  1. Rotate the tubes overnight at 4°C.
  2. Add 25 µL of washed and suspended Protein A/G magnetic beads to each IP sample.  
  3. Rotate the tube for 1 hour at 4°C.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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

  1. 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.
  2. Incubate the sample at 95°C for 15 minutes.
  3.  
  4. .  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.
  5. Incubate at 95°C for 10 minutes to deactivate Proteinase K.
  6. The DNA samples can now be purified with spin columns or by phenol/chloroform extraction.

 

DNA PCR Amplification

  1. 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.
  2. For the input control fraction only, dilute the template to 1% of the original concentration (1:100 dilution). All other samples are left undiluted.
  3. 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

  1. Perform real time PCR according to manufacturer’s recommendations for the SYBR reaction mix.

 

 

Protocol for Immunohistochemistry (IHC), Paraffin

 

Preparation - Perfusion and Paraffin Embedding

  1. Fix the tissue of interest by immersing it in 10% neutral buffered formalin (4% PFA-PBS) for 4-24 hours at room temperature. Fixation time and temperature depends on tissue type/size.
  2. Dehydrate by moving tissue through the following solutions twice for 30 minutes each:
    1. 70% Ethanol
    2. 95% Ethanol
    3. 100% Ethanol
    4. Xylene
  3. Embed the tissue in molten paraffin and after the paraffin solidifies, keep the blocks at 4˚C until sectioning.
  4. Use a microtome to cut the embedded tissue into 4-6 µm thick sections and float them in a 50°C water bath containing distilled water.
  5. Mount sections onto gelatin or poly-L-lysine coated slides and allow them to dry overnight. Slides can be safely stored at room temperature until ready for staining.

 

Immunofluorescent staining

  1. Deparaffinize and rehydrate by immersing the slides through the following solutions:
    1. Xylene: three times for 5 minutes each
    2. 100% Ethanol: twice for 5 minutes each
    3. 95% Ethanol: 5 minutes
    4. 70% Ethanol: 5 minutes
    5. 50% Ethanol: 5 minutes
    6. Distilled water: 5 minutes. Do not let the tissue dry from this point on.
  2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen or use rubber cement.
  3. For antigen retrieval using microwave, bring the slides to a boil in 10 mM sodium citrate buffer (pH 6.0) and then maintain at a sub-boiling temperature for 10 minutes. Thereafter, let it cool on bench-top for about 30 minutes and wash the sections by immersing them in distilled water for 5 minutes. 
  4. For permeablization of the tissue/cells, wash the sections twice for 10 minutes with 1% animal serum in PBS with 0.4% Triton X-100 (PBS-T). The species of the animal serum is dependent on the host of your secondary antibody (e.g. when using a goat anti-mouse secondary, use goat serum).
  5. Block any non-specific binding by incubating the tissue sections with 5% animal serum in PBS-T for 30 minutes at room temperature.
  6. Add primary antibody diluted in 1% animal serum PBS-T and incubate at room temperature for 1-2 hours followed by overnight at 4°C in humidified chamber. Use the recommended dilution of the antibody if specified on the datasheet and if not, then typical starting dilution can be 2-5 µg/ml.
  7. Wash sections twice with 1% serum PBS-T for 10 minutes each.
  8. Dilute secondary antibody in 1% serum PBS-T and incubate with sections at room temperature for 1-2 hours. Use the recommended dilution of the antibody as specified on the datasheet.
  9. Wash sections twice with 1% serum PBS-T for 10 minutes each.
  10. Optional: Double/Nuclear labeling
    1. Double labeling: If using a second primary antibody and appropriately matched secondary, repeat steps 5-8.
    2. Nuclear labeling: After application of all primary antibodies, DNA binding dyes such as DAPI can be applied, which are used without the need for secondary antibodies. Use the recommended dilution and incubation time as specified on the datasheet. After incubation, wash once for 5 minutes with PBS.
  11. Tap off excess wash and apply one drop of anti-fade mounting medium to the slide. Place a coverslip on the tissue sections. Ring the edges of the coverslip with clear fingernail polish to prevent the cells from drying. Allow nail polish to air dry.
  12. Slides may now be examined under a microscope with the appropriate fluorescent filter sets. Limiting the amount of time each slide is exposed to the microscope’s light aids in prolonging the signal and prevents photobleaching.
  13. Slides can be stored between -20°C and 4°C in a dark slide box or slide book.

 

Immunochromogenic Staining (ABC method with DAB)

  1. Deparaffinize and rehydrate by immersing the slides through the following wells:
    1. Xylene: three times for 5 minutes each
    2. 100% Ethanol: twice for 5 minutes each
    3. 95% Ethanol: 5 minutes
    4. 70% Ethanol: 5 minutes
    5. 50% Ethanol: 5 minutes
    6. Distilled water: 5 minutes. Do not let the tissue dry from this point on.
  2. Draw a circle on the slide around the tissue with a hydrophobic barrier pen or use rubber cement.
  3. For antigen retrieval using microwave, bring the slides to a boil in 10 mM sodium citrate buffer (pH 6.0) and then maintain at a sub-boiling temperature for 10 minutes. Thereafter, let it cool on bench-top for about 30 minutes and wash the sections by immersing them in distilled water for 5 minutes. 
  4. For blocking the endogenous peroxidase activity, quench the tissue sections with 3.0% hydrogen peroxide in methanol for at least 15 minutes. Afterwards, wash the sections by immersing them in distilled water for 5 minutes. 
  5. For permeablization of the tissue/cells, wash the sections twice for 10 minutes with 1% animal serum in PBS with 0.4% Triton X-100 (PBS-T). The species of the animal serum is dependent on the host of your secondary antibody. (e.g. when using a goat anti-mouse secondary, use goat serum).
  6. Block any non-specific binding by incubating the tissue sections with 5% animal serum in PBS-T for 30 minutes at room temperature.
  7. Add primary antibody diluted in 1% animal serum PBS-T and incubate at room temperature for 1-2 hours followed by overnight at 4°C in humidified chamber. Use the recommended dilution of the antibody if specified on the datasheet and if not, then typical starting dilution can be 2-5 µg/ml.
  8. Wash sections twice with 1% serum PBS-T for 10 minutes each.
  9. Add a biotinylated secondary antibody and incubate at room temperature for 1 hour. Use the recommended dilution of the antibody as specified on the datasheet
  10. Wash sections twice with 1% serum PBS-T for 10 minutes each.
  11. Add ABC-HRP reagent and incubate at room temperature for 1 hour. Follow manufacturer’s guidelines for reagent preparation.
  12. Wash sections twice in PBS for 10 minutes each.
  13. Prepare a working solution of DAB and apply to tissue sections. Monitor the reaction as the chromogenic reaction turns the epitope sites brown (time of color development may vary from few seconds to 10 minutes). Proceed to the next step when the intensity of the signal is appropriate for imaging. Important: DAB is a carcinogen! Always wear gloves and work in a fume hood when working with DAB. Deactivate and clean work area after use according to manufacturer’s instructions.
  14. Wash the sections twice in distilled water for 2 minutes each.
  15. To counterstain nuclei, use Hematoxylin according to the manufacturer’s instructions. Note: If you are using an aqueous chromogen instead of DAB (i.e. AEC, Fast Red, etc.), skip the following dehydration step and mount in aqueous media instead of organic mounting media.
  16. Dehydrate tissue sections by moving slides through the following solutions twice for 2 minutes each:
    1. 95% Ethanol
    2. 100% Ethanol
    3. Xylene
  17. Add mounting media to slides and top with coverslips. The DAB reaction is permanent and stable and can be analyzed under a brightfield microscope at any time.

More Resources:

Chromatin Immunoprecipitation (ChIP) Troubleshooting