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[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
作者: Nabila Aboulaich
1/20/2011, 10492 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.21

[Abstract]

[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).

材料与试剂

1.     蛋白酶抑制剂全片 (Roche)

2.     1kb DNA ladder (Invitrogen)

3.     Protein A- Protein G-琼脂微球(Santa Cruz Biotechnology)

4.     /氯仿/isoamylalchohol (Invitrogen)

5.     Pellet paint (Novagen)

6.     常用化学试剂(Sigma)

 

仪器与设备

1.     超声波细胞破碎仪

2.     摇床

3.     离心机

4.     PCR 

 

实验步骤

1.    70mg新鲜或者冷冻材料。用干净的刀片在4.5 mL预冷的PBS 缓冲液中将材料切成碎片,冰上进行此步骤。

2.    加入新配的HEPES/甲醛溶液,至终浓度为1%甲醛。 (加入0.5ml 11%HEPES-NaOH-甲醛溶液。一定要在实验前现用现配。)

3.    室温孵育10分钟。

4.    42000rpm离心5分钟。

5.    PBS缓冲液洗涤材料,离心,重复两次。

6.    加入CHIP溶菌缓冲液(70 mg 材料中加入1.6 mL,缓冲液中已加入蛋白酶抑制剂)。将样品分装至两个eppendorf离心管中,每个约800 uL

7.    将样品进行超声波破碎,以获得长度约为500-1200 bp的染色质。超声波破碎过程应全部在冰上进行。(参考程序:超声波处理2秒,暂停10秒,6个循环。)

8.    高速离心超声波破碎产物。合并每个样品的两个离心管中的产物。

9.    5 uL样品,在1%琼脂糖胶中电泳检测大小。若条带大小约为3kb,则可进行下面的步骤。(通常未解交联的染色质电泳的条带大于3kb

10. 取一部分样品作为PCRinput对照。(5-20%

11. 预纯化染色质:使用树脂,如Protein A,在免疫沉淀反应后得到免疫复合物。每管40uL分装的预洗涤Protein A琼脂柱并加入0.5 X RIPA 缓冲液 +蛋白酶抑制剂稀释样品。根据电泳结果,加入大约100-200uL超声波破碎产物。预纯化混合物总体积为1ml4,翻转摇床孵育1小时以上。预纯化的目的是去除能结合在树脂上的非特异杂质。将一个样品平均分至多管预纯化的效果要好于将一个样品预纯化后再分装至多管。短暂离心(2000 rpm, 1 min),小心将上清转移至新的离心管中。加入适当的抗体(约3ug,取决于抗体浓度),4,翻转摇床孵育3小时或过夜。

12. 将免疫沉淀下的DNA-蛋白复合物加入新的预洗涤过的琼脂柱,4孵育1.5小时。

13. 短暂离心(2000 rpm 1 min)复合物。用800ul,0.5 X RIPA缓冲液洗涤沉淀,重复3次。(加入缓冲液,振荡离心管混匀,短暂离心,去除上清,重复洗涤)

14. 加入300ul digesting缓冲液,65孵育4小时-过夜,解除交联。

15. 每管加入等体积酚/氯仿/ isoamylalchohol,涡旋振荡,并4高速离心10分钟,转移液态上清至新的离心管,乙醇沉淀DNA。因为得到的DNA量可能会很少,所以建议用糖原或pellet paint作为载体。

16. 4,最高速离心30分钟,去除上清,干燥沉淀,用20ul水重悬沉淀。用此作为PCR的模板,每个反应用2ul

 

配方

1.     10XHEPES-NaOH 缓冲液

1M Hepes pH 7.8

11%甲醇溶液 (approx)

交联时工作液浓度为1X

此溶液为10X母液,实验时应现用现配,工作液浓度为1X

2.     ChIP 溶菌缓冲液:

50 mM Tris-Cl pH 8.0

10 mM EDTA

1% SDS

+蛋白酶抑制剂

3.     RIPA 缓冲液 (1X)

10 mM Tris-Cl pH 8.0

1 mM EDTA

0.5 mM EGTA

140 mM NaCl

1 % Triton X 100

0.1 % 脱氧胆酸钠

0.1 % SDS

+ 蛋白酶抑制剂

4.     Digesting 缓冲液

mM Tris-Cl pH 8.0

1 mM EDTA

100 mM NaCl

0.5 % SDS

(一些人喜欢加入100 ug/mL K蛋白酶, 但是当将样品加热至65和开始进行酚/氯仿抽提实验时,K蛋白酶将不再起作用).

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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



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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.

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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 ....

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