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Chromatin Immunoprecipitation (ChIP) Protocol from Tissue   

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Materials and Reagents

  1. Frozen tissue
  2. Protease inhibitors complete mini pill (F. Hoffmann-La Roche)
  3. 1 kb DNA ladder (Life Technologies, Invitrogen™)
  4. Protein A- or protein G- agarose (Santa Cruz Biotechnology)
  5. Phenol-chloroform-isoamylalchohol (Life Technologies, Invitrogen™)
  6. Pellet paint (Novagen)
  7. General chemicals (Sigma-Aldrich)
  8. PBS
  9. Hepes
  10. Tris-HCl (pH 8.0)
  11. EDTA
  12. SDS
  13. EGTA
  14. NaCl
  15. Triton X-100
  16. Sodium deoxycholate
  17. Agarose gel
  18. HEPES-NaOH buffer (see Recipes)
  19. ChIP lysis buffer (see Recipes)
  20. RIPA buffer (see Recipes)
  21. Digesting buffer (see Recipes)


  1. Centrifuges
  2. Sonicator
  3. Razor blade
  4. Shaker
  5. PCR machine


  1. Start with 70 mg of fresh or frozen tissue. Mince the tissue using a clean razor blade in 4.5 ml of ice cold PBS on ice into small pieces.
  2. Add freshly made HEPES-formaldehyde solution to a final concentration of 1% formaldehyde. (0.5 ml of 11% formaldehyde in HEPES-NaOH. Make up this solution right before use, enough for one use only).
  3. Incubate with mixing at room temperature for 10 min.
  4. Centrifuge the cross-linked tissue at 2,000 rpm for 5 min, 4 °C.
  5. Wash the tissue in PBS and centrifuge again (twice).
  6. Add ChIP lysis buffer (1.6 ml for 70 mg of starting tissue) with protease inhibitors. Split the sample into two eppendorf tubes, 800 μl each and make sure there is roughly the same amount of tissue in each.
  7.  Sonicate the tissue (ON ICE at all times) to obtain chromatin with an average shear size of 500-1200 bp. (With our lab sonicator this is achieved by sonicating at setting 2, 10 sec per cycle, 6 cycles total).
  8. Centrifuge the sonicated chromatin at high speed and pellet the debris from sonication. Pool the two tubes of chromatin for each sample.
  9. Run out 5 μl of chromatin on a 1% agarose gel with a 1 kb DNA ladder to determine shear size. If the size is close to 3 kb, proceed with ChIPs (Chromatin with un-reversed crosslinks usually runs slower on a gel and appears larger than the actual size).
  10. Save a small aliquot as input control for PCR (5-20%).
  11. Pre-clear Chromatin: For this step, use the resin that you intend to use to capture immune complexes after the immunoprecipitation, such as Protein A agarose. Dilute the chromatin in 0.5x RIPA buffer with protease inhibitors and add to 40 μl packed, pre-washed Protein A agarose. Out of 1600 μl of sonicated chromatin, I use about 100-200 μl per IP (depending on how much you see on the gel). Total volume of preclearing mix is 1 ml. Incubate for 1 h or more with rocking at 4 °C. The purpose of pre-clearing is to remove material in the chromatin that binds non-specifically to the resin. Use one pre-clearing tube for each IP rather than pre-clearing concentrated chromatin in one tube. Spin down the pre-cleared chromatin (2,000 rpm, 1 min) and carefully transfer the supernatant to a fresh tube. Add the appropriate antibody to the IP (3 μg, but it depends on antibody) and incubate for 3 h (or overnight) at 4 °C, with rocking.
  12. After immunoprecipitating the protein-DNA complexes, add the IP mixture to fresh pre-washed agarose beads/resin and incubate for 1.5 h at 4 °C.
  13. Spin the IPs at 2,000 rpm for 1 min and remove the supernatant. Wash IPs 3 times with 800 μl of 0.5x RIPA buffer (add the buffer, shake the tubes well and then spin down the resin each time, remove supernatant and repeat wash).
  14. Add 300 μl of digesting buffer and incubate the tubes at 65 °C for a minimum of 4 h, max overnight to reverse the crosslinks.
  15. Add an equal volume of phenol-chloroform-isoamylalchohol to the tubes, vortex and centrifuge at high speed for 10 min, 4 °C. Transfer the aqueous supernatant to a fresh tube and ethanol precipitate the DNA. Since the amount of DNA recovered is very little, it is recommended that you use a carrier for precipitation, such as glycogen or pellet paint.
  16. Centrifuge ethanol-precipitated samples at top speed for 30 min at 4 °C and remove the supernatant. Dry the DNA pellet and resuspend in 20 μl of water. Use this DNA for PCR amplification, 2 μl per reaction.


  1. HEPES-NaOH Buffer
    1 M Hepes pH 7.8
    Formaldehyde concentration 11% (approx)
    Use one-tenth volume to crosslink
    * make up the HEPES-NaOH but add formaldehyde only before use. This is a 10x stock.
  2. ChIP Lysis Buffer:
    50 mM Tris-Cl pH 8.0
    10 mM EDTA
    1% SDS
    + protease inhibitors
  3. RIPA Buffer (1x)
    10 mM Tris-Cl pH 8.0
    1 mM EDTA
    0.5 mM EGTA
    140 mM NaCl
    1 % Triton X 100
    0.1 % Sodium Deoxycholate
    0.1 % SDS
    + protease inhibitors
  4. Digesting Buffer
    50 mM Tris-Cl pH 8.0
    1 mM EDTA
    100 mM NaCl
    0.5 % SDS
    (some people like to add 100 μg/ml proteinase K, but since you are going to heat the sample at 65 °C, and then do a phenol chloroform extraction, it’s not really necessary).
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Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Aboulaich, N. (2011). Chromatin Immunoprecipitation (ChIP) Protocol from Tissue. Bio-101: e21. DOI: 10.21769/BioProtoc.21.
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If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Rahul kumar
Hi sir i am work on chip from tissue problem is that after sonication we get two fraction one is pellet and another is supernatant then confusion is that which portion contain DNA fragment.
1/21/2015 2:13:19 AM Reply
vikas sharma
Hello sir,

i am also trying to do chip on tissue samples , initially the amount of sample taken by me was 70 mg. after sonication() step i took around 100microlitre of this sonicated product and followed the phenol-CHCL3(ph 7.6-8 which is used to isolate dna ) procedure to isolate the dna. the O.D obtained was 45ng/microlitre and 260/280 ratio was 2.1(i know this ratio represents RNA ) i also checked my product on 2 % agarose gel but found no band in my products.
so what does it conclude...
where my dna has gone....has it been degraded... or was present in the pellet of cells that i have thrown as waste after centrifugation... or was present in a different layer while dna purification that was removed by me...
if i can isolate rna(which gets easily degraded due to RNase action ) then how could i not get dna as dna is more stable than rna and as all hygienic conditions were maintained .
needs your suggestion to this situation.

thanks for reading with all your patience ....
9/19/2011 11:02:42 AM Reply
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