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[Bio101] ChIP Assay for Cell Culture
[Bio101] 对培养细胞的染色体免疫共沉淀分析   

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Abstract

Chromatin immunoprecipitation (ChIP) is a method used to determine the location of DNA binding sites on the genome for a particular protein of interest. Following crosslinking, cells are lysed and the DNA is broken into pieces 0.2-1.0 kb in length by sonication. At this point immunoprecipitation is performed resulting in the purification of protein–DNA complexes. The identity and quantity of the isolated DNA fragments can then be determined by PCR. This protocol describes how to perform a ChIP experiment and can be applied to different types of cell culture.

Keywords: ChIP(染色质免疫沉淀技术), Immunoprecipitation(免疫共沉淀), DNA(DNA)

Materials and Reagents

  1. Salmon Sperm DNA (Life Technologies, InvitrogenTM, catalog number: AM9680 )
  2. Protein inhibitors
    1. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: 78830-5G )
    2. Aprotinin (Sigma-Aldrich, catalog number: A3428-10MG )
    3. Pepstatin A (Sigma-Aldrich, catalog number: P5318-5MG )
  3. Formaldehyde (Thermo Fisher Scientific, catalog number: F75F-1GAL )
  4. Protein A agarose beads (Upstate, Millipore, catalog number: 16-157 )
  5. General chemicals (Sigma-Aldrich)
  6. DPBS (Life Technologies, InvitrogenTM, catalog number: 14190-250 )
  7. QIAGEN PCR purification kit (QIAGEN, catalog number: 28104 )
  8. ChIP lysis buffer (see Recipes)
  9. Low salt wash buffer (see Recipes)
  10. High salt wash buffer (see Recipes)
  11. LiCl buffer (see Recipes)
  12. TE buffer (see Recipes)
  13. Elution buffer (see Recipes)

Equipment

  1. Standard bench-top refrigerated centrifuge that can reach at least 13,000 rpm
  2. Conical tube
  3. Rotating platform

Procedure

  1. Grow about 1-2 x 107 cells in a 100 mm petri dish, and then crosslink by adding formaldehyde to a final concentration of 1%. Incubate at 37 °C for 10 min.
  2. Wash cells twice using ice cold DPBS containing protease inhibitors [1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstatin A].
    Note: Add protease inhibitors to the DPBS just prior to use. PMSF has a half-life of approximately 30 min in aqueous solutions.
  3. Scrape cells in 2 ml ice cold DPBS containing the protease inhibitors and transfer into a conical tube, then pellet cells for 4 min at 2,000 rpm at 4 °C.
  4. Remove the PBS and resuspend cells in ChIP lysis buffer with protease inhibitors (inhibitors: 1 mM PMSF, 1 μg/ml aprotinin and 1 μg/ml pepstatin A), then incubate for 10 min on ice.
  5. Sonicate the cells to shear DNA to lengths between 200 and 1,000 bp; bubbles will be produced, so spin down to get rid of bubbles and then repeat the sonication step 5 times, 1 min each time, being sure to keep samples ice cold.
  6. Centrifuge the samples for 10 min at 13,000 rpm at 4 °C, and determine the OD260 of the lysates.
  7. Dilute the sonicated lysates to OD260 2 with ChIP dilution buffer. Add 60 μl Protein A agarose beads to the sonicated lysates and rotate at 4 °C for 1 h to reduce non-specific binding.
  8. Pellet agarose by brief centrifugation and collect the supernatant fraction, 20 μl of lysates should be taken out as input control.
  9. Add the immunoprecipitating antibody (the amount will vary per antibody) to the 2 ml supernatant fraction and incubate for 2 h at 4 °C with rotation. For negative control, perform a no-antibody immunoprecipitation by incubating the supernatant fraction with 60 μl mix of 0.1 μg/μl salmon sperm DNA and Protein A agarose beads for one hour at 4 °C with rotation.
  10. Pellet agarose by gentle centrifugation (700 to 1,000 rpm at 4 °C, ~1 min). Carefully remove the supernatant that contains unbound, non-specific DNA. Wash the Protein A agarose/antibody/histone complex for 3-5 min on a rotating platform with 1 ml of each of the buffers listed in the order as given: Low salt wash buffer; high salt wash buffer; LiCl buffer; TE buffer.
  11. Elute the histone complex from the antibody by adding 250 μl elution buffer to the pelleted Protein A agarose/antibody/histone complex from step 10 above. Vortex briefly to mix and incubate at room temperature for 15 min with rotation. Spin down agarose, and carefully transfer the supernatant fraction (eluate) to a separate clean tube and repeat elution. Combine eluates (total volume = approximately 500 μl).
  12. Add 20 μl 5 M NaCl to the combined eluates (500 μl) and reverse histone-DNA crosslinks by heating at 65 °C for 4 h.
    Note: Include the input DNA from this step.
  13. Dilute with 2 ml TE buffer, add 5 μl of ice acetic acid, purify the DNA by using the QIAGEN PCR purification kit, then recover DNA in 50 μl elution buffer.
  14. Perform PCR and standard agarose gel electrophoresis. PCR conditions must be determined empirically.

Recipes

  1. ChIP lysis buffer
    1% SDS
    10 mM EDTA
    50 mM Tris-HCl (pH 8.0)
  2. Low salt wash buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.0)
    150 mM NaCl
  3. High salt wash buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 8.0)
    500 mM NaCl
  4. LiCl buffer
    0.25 M LiCl
    1% NP-40
    1% SDC
    1 mM EDTA
    10 mM Tris-HCl (pH 8.0)
  5. TE buffer
    20 mM Tris-HCl (pH 8.0)
    1 mM EDTA (pH 8.0)
  6. Elution buffer
    1% SDS
    0.1 M NaHCO3

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. The protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA.

References

  1. Chen, R., Liliental, J. E., Kowalski, P. E., Lu, Q. and Cohen, S. N. (2011). Regulation of transcription of hypoxia-inducible factor-1alpha (HIF-1alpha) by heat shock factors HSF2 and HSF4. Oncogene 30(22): 2570-2580.

简介

染色质免疫沉淀(ChIP)是用于确定特定目标蛋白质的基因组上的DNA结合位点的位置的方法。 交联后,裂解细胞,通过超声处理将DNA断裂成长度为0.2-1.0kb的片段。 此时进行免疫沉淀,从而纯化蛋白质-DNA复合物。 然后可以通过PCR测定分离的DNA片段的身份和数量。 该协议描述了如何进行ChIP实验,并可以应用于不同类型的细胞培养。

关键字:染色质免疫沉淀技术, 免疫共沉淀, DNA

材料和试剂

  1. 鲑精DNA(Life Technologies,Invitrogen TM,目录号:AM9680)
  2. 蛋白抑制剂
    1. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:78830-5G)
    2. 抑肽酶(Sigma-Aldrich,目录号:A3428-10MG)
    3. 胃酶抑素A(Sigma-Aldrich,目录号:P5318-5MG)
  3. 甲醛(Thermo Fisher Scientific,目录号:F75F-1GAL)
  4. 蛋白A琼脂糖珠(Upstate,Millipore,目录号:16-157)
  5. 一般化学品(Sigma-Aldrich)
  6. DPBS(Life Technologies,Invitrogen TM,目录号:14190-250)
  7. QIAGEN PCR纯化试剂盒(QIAGEN,目录号:28104)
  8. ChIP裂解缓冲液(参见配方)
  9. 低盐洗涤缓冲液(见配方)
  10. 高盐洗涤缓冲液(见配方)
  11. LiCl缓冲液(见配方)
  12. TE缓冲区(参见配方)
  13. 洗脱缓冲液(见配方)

设备

  1. 标准台式冷冻离心机,可至少达到13,000转/分
  2. 圆锥管
  3. 旋转平台

程序

  1. 在100mm培养皿中生长约1-2×10 7个细胞,然后通过加入甲醛至终浓度为1%进行交联。 在37℃孵育10分钟。
  2. 使用含有蛋白酶抑制剂[1mM PMSF,1μg/ml抑肽酶和1μg/ml胃蛋白酶抑制剂A]的冰冷DPBS洗涤细胞两次。
    注意:在使用前向DPBS中加入蛋白酶抑制剂。 PMSF在水溶液中的半衰期约为30分钟。
  3. 刮擦细胞在含有蛋白酶抑制剂的2ml冰冷的DPBS中,并转移到锥形管中,然后在4℃下以2,000rpm沉淀细胞4分钟。
  4. 取出PBS,并用蛋白酶抑制剂(抑制剂:1mM PMSF,1μg/ml抑肽酶和1μg/ml胃蛋白酶抑制剂A)在ChIP裂解缓冲液中重悬细胞,然后在冰上孵育10分钟。
  5. 超声处理细胞以剪切DNA长度在200和1000 bp之间;会产生气泡,因此旋下去除气泡,然后重复超声步骤5次,每次1分钟,一定要保持样品冰冷。
  6. 在4℃下以13,000rpm离心样品10分钟,并测定裂解物的OD 260。
  7. 用ChIP稀释缓冲液将经超声处理的裂解物稀释至OD 260。加入60μl蛋白A琼脂糖珠到超声处理裂解物,并在4℃下旋转1小时,以减少非特异性结合。
  8. 通过短暂离心沉淀颗粒琼脂糖并收集上清液部分,应当取出20μl裂解物作为输入对照。
  9. 加入免疫沉淀抗体(每次抗体的量将不同)到2ml上清液部分中,并在4℃下旋转孵育2小时。对于阴性对照,通过将上清液部分与0.1μl/μl鲑鱼精子DNA和蛋白A琼脂糖珠的60μl混合物在4℃下旋转孵育1小时来进行无抗体免疫沉淀。
  10. 通过温和离心(700至1,000rpm,4℃,〜1分钟)沉淀颗粒琼脂糖。小心地去除含有未结合的非特异性DNA的上清液。在旋转平台上洗涤蛋白A琼脂糖/抗体/组蛋白复合物3-5分钟,其中按照给定顺序列出的每种缓冲液1ml:低盐洗涤缓冲液;高盐洗涤缓冲液; LiCl缓冲液; TE缓冲液。
  11. 通过向来自上述步骤10的沉淀的蛋白A琼脂糖/抗体/组蛋白复合物中加入250μl洗脱缓冲液,从抗体洗脱组蛋白复合物。短暂涡旋混合并在室温下孵育15分钟,旋转。旋转琼脂糖,并小心转移上清部分(洗脱液)到一个单独的干净管,并重复洗脱。合并洗脱液(总体积=约500μl)。
  12. 通过在65℃加热4小时,向合并的洗脱液(500μl)和反向组蛋白-DNA交联中加入20μl5M NaCl。
    注意:包括此步骤的输入DNA。
  13. 用2ml TE缓冲液稀释,加入5μl冰醋酸,使用QIAGEN PCR纯化试剂盒纯化DNA,然后在50μl洗脱缓冲液中回收DNA。
  14. 进行PCR和标准琼脂糖凝胶电泳。 PCR条件必须根据经验确定。

食谱

  1. ChIP裂解缓冲液
    1%SDS
    10 mM EDTA
    50mM Tris-HCl(pH8.0)
  2. 低盐洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.0) 150mM NaCl
  3. 高盐洗涤缓冲液
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH8.0) 500 mM NaCl
  4. LiCl缓冲液
    0.25 M LiCl
    1%NP-40
    1%SDC
    1mM EDTA
    10mM Tris-HCl(pH8.0)
  5. TE缓冲区
    20mM Tris-HCl(pH8.0) 1mM EDTA(pH8.0)
  6. 洗脱缓冲液
    1%SDS
    0.1 M NaHCO 3 3/v/v

致谢

这项工作由杭州高新区5050项目,杭州市海外退学基金资助,浙江省ZJ1000项目资助。 该协议是在美国加利福尼亚州斯坦福大学遗传学系Cohen实验室开发的。

参考文献

  1. Chen,R.,Liliental,J.E.,Kowalski,P.E.,Lu,Q.and Cohen,S.N。(2011)。 缺氧诱导因子-1alpha(HIF-1alpha)通过热休克因子HSF2和 HSF4。 Oncogene 30(22):2570-2580。
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引用:Chen, R. (2011). ChIP Assay for Cell Culture. Bio-protocol Bio101: e23. DOI: 10.21769/BioProtoc.23;
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