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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   

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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).

简介

材料和试剂

  1. 蛋白酶抑制剂完全迷你丸(Roche)
  2. 1 kb DNA ladder(Invitrogen)
  3. 蛋白A-或蛋白G-琼脂糖(Santa Cruz Biotechnology)
  4. 苯酚 - 氯仿 - 异戊醇(Invitrogen)
  5. 颗粒漆(Novagen)
  6. 一般化学品(Sigma)

设备

  1. 超声波仪
  2. 振动器
  3. 离心机
  4. PCR机

程序

  1. 开始与70毫克新鲜或冷冻的组织。 使用干净的剃刀刀片将4.5ml冰冷的PBS中的组织在冰上切碎成小块
  2. 加入新鲜制备的HEPES-甲醛溶液至最终浓度为1%甲醛。 (0.5ml 11%甲醛的HEPES-NaOH溶液,在使用前配制该溶液,仅供一次使用)。
  3. 在室温下混合孵育10分钟
  4. 在4℃下,以2,000rpm离心交联的组织5分钟
  5. 在PBS中洗涤组织,并再次离心(两次)
  6. 加入蛋白酶抑制剂的ChIP裂解缓冲液(对于70mg起始组织1.6ml)。将样品分成两个eppendorf管,每个800微升,并确保每个大致相同数量的组织。
  7.  对组织(ON ICE)进行超声处理,以获得平均剪切大小为500-1200 bp的染色质。 (使用我们的实验室超声波仪,这是通过在设置2,每个周期10秒,共6个周期的声波处理实现的)。
  8. 以高速离心超声处理的染色质并从超声处理中沉淀碎片。合并每个样品的两个染色质管
  9. 运行5μl的染色质在1%琼脂糖凝胶与1kb DNA梯子,以确定剪切大小。如果大小接近3 kb,继续ChIPs(具有未反转交联的染色质通常在凝胶上运行较慢,并且显示大于实际大小)。
  10. 保存一小份作为PCR的输入对照(5-20​​%)。
  11. 预清除染色质:对于此步骤,使用您打算用于捕获免疫沉淀后的免疫复合物的树脂,如蛋白A琼脂糖。用蛋白酶抑制剂稀释0.5x RIPA缓冲液中的染色质,加入40μl包装的,预洗的蛋白A琼脂糖。在1600μl超声处理的染色质中,我使用大约100-200μl每IP(取决于你在凝胶上看到多少)。预清除混合物的总体积为1ml。在4℃摇动下孵育1小时或更长时间。预清除的目的是去除与树脂非特异性结合的染色质中的材料。每个IP使用一个预清洗管,而不是预先清除浓缩染色质在一个管。旋转预清除的染色质(2,000 rpm,1分钟),小心转移上清液到一个新的管。向IP中加入适当的抗体(3μg,但它取决于抗体),并在4℃下摇动孵育3小时(或过夜)。
  12. 免疫沉淀蛋白-DNA复合物后,将IP混合物加入新鲜的预洗涤的琼脂糖珠/树脂中,并在4℃下孵育1.5小时。
  13. 旋转IPs在2000 rpm下1分钟,并去除上清液。用800μl0.5x RIPA缓冲液洗涤IP3次(加入缓冲液,摇匀管,然后每次旋转树脂,除去上清液并重复洗涤)。
  14. 加入300μl消化缓冲液,并将管在65°C孵育至少4小时,最大过夜,以扭转交联。
  15. 向管中加入等体积的苯酚 - 氯仿 - 异戊醇,涡旋并在4℃高速离心10分钟。将含水上清液转移到新管中,乙醇沉淀DNA。由于回收的DNA量很少,因此建议您使用载体进行沉淀,如糖原或颗粒漆。
  16. 离心乙醇沉淀的样品在最高速度30分钟,在4℃,并去除上清液。 干燥DNA沉淀并重悬于20μl水中。 使用此DNA进行PCR扩增,每个反应2μl。

食谱

  1. HEPES-NaOH缓冲液
    1 M Hepes pH 7.8
    甲醛浓度11%(约)
    使用十分之一体积交联
    *补充HEPES-NaOH,但在使用前只加入甲醛。 这是10x股票。
  2. ChIP裂解缓冲液:
    50mM Tris-Cl pH8.0
    10 mM EDTA
    1%SDS
    +蛋白酶抑制剂
  3. RIPA缓冲区(1x)
    10mM Tris-Cl pH8.0
    1mM EDTA
    0.5mM EGTA 140mM NaCl 1%Triton X 100
    0.1%脱氧胆酸钠
    0.1%SDS
    +蛋白酶抑制剂
  4. 摘要缓冲区
    50mM Tris-Cl pH8.0
    1mM EDTA
    100 mM NaCl
    0.5%SDS
    (有些人喜欢添加100μg/ml蛋白酶K,但由于你要在65°C加热样品,然后进行苯酚氯仿萃取,这不是真的必要)。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Aboulaich, N. (2011). Chromatin Immunoprecipitation (ChIP) Protocol from Tissue. Bio-protocol Bio101: e21. DOI: 10.21769/BioProtoc.21;
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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.
1/21/2015 2:13:19 AM Reply
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 ....
9/19/2011 11:02:42 AM Reply