欢迎您, 登录 | 注册

首页 | English

X
加载中

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Chromatin Immunoprecipitation (ChIP) Protocol from Tissue
[Bio101] 组织的染色体免疫共沉淀实验方案

分子生物学 > DNA > DNA-蛋白质相互作用

[Abstract]

Materials and Reagents

  1. Protease inhibitors complete mini pill (Roche)
  2. 1 kb DNA ladder (Invitrogen)
  3. Protein A- or Protein G-agarose (Santa Cruz Biotechnology)
  4. Phenol-chloroform-isoamylalchohol (Invitrogen)
  5. Pellet paint (Novagen)
  6. General chemicals (Sigma)

Equipment

  1. Sonicator
  2. Shaker
  3. Centrifuge
  4. PCR machine

Procedure

  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.

Recipes

  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).


How to cite this protocol: Aboulaich, N. (2011). Chromatin Immunoprecipitation (ChIP) Protocol from Tissue. Bio-protocol Bio101: e21. DOI: 10.21769/BioProtoc.21; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
1/21/2015 2:13:19 AM  

Rahul kumar
DIPAS

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.
Thanks.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

9/19/2011 11:02:42 AM  

vikas sharma
AIIMS

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 ....

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register