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Immunoprecipitation for Cell Culture
培养细胞的免疫沉淀   

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Abstract

Immunoprecipitation (IP) is a method to pull down a protein out of solution using an antibody that specifically binds to that particular protein. Immunoprecipitation is a powerful technique to isolate and concentrate a particular protein from a sample containing many thousands of different proteins, to test protein-protein interactions, and to pull multiple members of a complex out of solution by latching onto one member with an antibody. This protocol describes a general immunoprecipitation strategy using cell cultures as starting material.

Keywords: Immunofluorscence (免疫荧光), Cells (细胞), Localization (定位), Fluorescence microscope (荧光显微镜)

Materials and Reagents

  1. HeLa S3 and HeLa cells were cultured in Dulbecco’s modified Eagles Medium containing 10% fetal bovine serum (Invitrogen) and antibiotics
  2. Protein A beads (Bio-Rad Laboratories); Protein G beads (Santa Cruz Biotechnology)
    Note: For rabbit polyclonal antibodies and mouse monoclonal antibodies IgG2a, IgG2b, IgG3, we usually couple antibodies to protein A beads; For mouse monoclonal antibodies IgG1, rat monoclonal antibodies, and mouse, rat, goat, and donkey polyclonal antibodies, we usually couple antibodies to protein G beads.
  3. Bifunctional cross-linker dimethyl pimelimidate (DMP) (MW: 259.177) (Sigma-Aldrich, catalog number: D8388 )
  4. Phosphate buffered saline (PBS)
  5. Tween 20
  6. Triton X-100
  7. Sodium borate (pH 9.0)
  8. Hepes
  9. KCl
  10. NaN3
  11. NP-40
  12. Glycerol
  13. EGTA
  14. MgCl2
  15. DTT
  16. Microcystin
  17. Leupeptin
  18. Pepstatin
  19. Chymostatin
  20. β-mercaptoethanol
  21. Bromophenol blue
  22. PBST buffer (see Recipes)
  23. Lysis buffer (see Recipes)
  24. 1x SDS protein gel sample loading buffer (see Recipes)

Equipment

  1. Hematology/chemistry mixer (Fisher Scientific)
  2. Centrifuge (Eppendorf 5415D centrifuge)
  3. Incubator

Procedure

  1. Coupling antibody to Protein A beads
    1. Wash 30 μl beads with 20 fold volume (20 vol) PBS or PBST twice.
      Notes:
      1. For washing beads, we usually add buffer to the beads, mix in the tube several times, spin down the beads, and remove the supernatant.
      2. Spin down the beads at 4,000 rpm, 30 sec.
      3. We usually couple 1 μg antibody with 3 μl beads. If beads are stored in 1: 1 storing buffer, thus take 6 μl of the suspension of beads and buffer.
    2. Dilute 10 μg antibody in 100 μl PBS.
    3. Add diluted antibody to beads, rotate on the Hematology/chemistry mixer equipment for 1 h at room temperature (RT).
    4. Spin down beads, wash and resuspend in 20 vol 0.2 M sodium borate (pH 9.0).
    5. Add 20 mM DMP to crosslink the antibody to the beads.
      Note: We usually take dry DMP powder directly (DMP powder is stored at 4 °C) and add 5.2 mg DMP per 1 ml total sodium borate suspension.
    6. Incubate DMP in the sodium borate for 30 min, rotate, RT.
    7. Spin down the bead and wash once with 20 vol 1 M Tris-HCl (pH 7.7).
    8. Spin down the bead and resuspend in 20 vol 1 M Tris-HCl (pH 7.7). Incubate for 2 h, rotate, RT, to quench the activity of DMP.
    9. Spin down beads and wash with PBS twice.
    10. Spin down the beads and store antibody coupled beads in 60 μl PBS (33% beads), add 0.05% NaN3. Store the beads at 4 °C for a couple of months.

  2. Lysis of cells
    1. Lyse the cells in 7 vol lysis buffer.
    2. Put the lysis solution on ice for 30 min to 1 h.
    3. Centrifuge the lysis solution at 13,000 rpm for 10 min, 4 °C.
    4. Transfer supernatants. The supernatants can be stores at -80 °C for future use.

  3. Coimmunoprecipitation
    1. Incubate 3 μl antibody coupled beads in 250 μl cell lysis supernatants, rotate overnight at 4 °C.
    2. Wash beads 4 times with 20 vol lysis buffer containing 500 mM KCl and 0.5 % NP-40, and wash once with lysis buffer.
      Note: For wash beads, we usually add buffer to the beads, mix the tube several times, spin down the beads, and remove the supernatant.
    3. Elute protein with 1x SDS protein gel sample loading buffer, separated by SDS-PAGE (5-15%) and analyze by western blot.

Recipes

  1. PBST buffer
    PBS plus 0.1 % Tween 20 or 0.1 % Triton X-100
  2. Lysis buffer
    50 mM Hepes (pH 7.4)
    200 mM KCl
    0.3% NP-40
    10% glycerol
    1 mM EGTA
    1 mM MgCl2
    0.5 mM DTT
    0.5 μM microcystin
    10 μg ml-1 each of leupeptin, pepstatin, and chymostatin
  3. 1x SDS protein gel sample loading buffer
    50 mM Tris-HCl (pH 6.8)
    2% SDS
    10% glycerol
    1% β-mercaptoethanol
    12.5 mM EDTA
    0.02 % bromophenol blue

Acknowledgments

This protocol was modified from an immunoprecipitation protocol developed in the laboratory of Dr. Guowei Fang (Department of Biology, Stanford University, Stanford, CA, USA). This work was supported by a Burroughs-Wellcome Career Award in Biomedical Research (G.F.) and by grants from National Institutes of Health (GM062852 to G.F.).

References

  1. Zhu, H., Coppinger, J. A., Jang, C. Y., Yates, J. R., 3rd and Fang, G. (2008). FAM29A promotes microtubule amplification via recruitment of the NEDD1-gamma-tubulin complex to the mitotic spindle. J Cell Biol 183(5): 835-848.

简介

免疫沉淀(IP)是使用特异性结合该特定蛋白的抗体将蛋白从溶液中取出的方法。 免疫沉淀是从含有数千种不同蛋白质的样品中分离和集中特定蛋白质,测试蛋白质 - 蛋白质相互作用,以及通过用抗体锁定到一个成员上而将复合物的多个成员从溶液中抽出的强大技术。 该协议描述了使用细胞培养物作为起始材料的一般免疫沉淀策略。

关键字:免疫荧光, 细胞, 定位, 荧光显微镜

材料和试剂

  1. HeLa S3和HeLa细胞在含有10%胎牛血清(Invitrogen)和抗生素的Dulbecco's modified Eagles Medium中培养。
  2. 蛋白A珠(Bio-Rad Laboratories); 蛋白G珠子(Santa Cruz Biotechnology)
    注意:对于兔多克隆抗体和小鼠单克隆抗体IgG2a,IgG2b,IgG3,我们通常将抗体偶联至蛋白A珠; 对于小鼠单克隆抗体IgG1,大鼠单克隆抗体和小鼠,大鼠,山羊和驴多克隆抗体,我们通常将抗体与蛋白G珠偶联。
  3. 双功能交联剂二甲基庚二亚胺二甲酯(DMP)(MW:259.177)(Sigma-Aldrich,目录号:D8388)
  4. 磷酸盐缓冲盐水(PBS)
  5. 吐温20
  6. Triton X-100
  7. 硼酸钠(pH9.0)
  8. Hepes
  9. KCl
  10. NaN 3
  11. NP-40
  12. 甘油
  13. EGTA
  14. MgCl 2
  15. DTT
  16. 微囊藻毒素
  17. 亮肽素
  18. 胃蛋白酶
  19. 抑菌素
  20. β-巯基乙醇
  21. 溴酚蓝
  22. PBST缓冲区(参见配方)
  23. 裂解缓冲液(见配方)
  24. 1x SDS蛋白凝胶样品上样缓冲液(见配方)

设备

  1. 血液学/化学混合器(Fisher Scientific)
  2. 离心机(Eppendorf 5415D离心机)
  3. 孵化器

程序

  1. 将抗体偶联至蛋白A珠
    1. 用20倍体积(20体积)PBS或PBST洗涤30μl珠两次 注意:
      1. 对于洗涤珠子,我们通常向珠子中加入缓冲液,在管中混合几次,将珠子旋转下来,并除去上清液。
      2. 以4000转/分,30秒的速度旋转珠子。
      3. 我们通常将1μg抗体与3μl珠偶联。 如果珠子存储在1:1的储存缓冲液中,则需要取6μl的珠子和缓冲液悬浮液。
    2. 在100μlPBS中稀释10μg抗体
    3. 向珠子中加入稀释的抗体,在室温(RT)下在血液/化学混合器设备上旋转1小时
    4. 旋转珠,洗涤并重悬于20体积0.2M硼酸钠(pH9.0)中
    5. 加入20mM DMP以使抗体与珠子交联 注意:我们通常直接使用干燥的DMP粉末(DMP粉末储存在4℃),并且每1ml总硼酸钠悬浮液添加5.2mg DMP。
    6. 在硼酸钠中孵育DMP 30分钟,旋转,RT
    7. 旋转小珠,并用20体积1M Tris-HCl(pH7.7)洗涤一次
    8. 旋转珠子并重悬于20体积的1M Tris-HCl(pH7.7)中。 孵育2小时,旋转,RT,以淬灭DMP的活性
    9. 旋转珠并用PBS洗涤两次
    10. 旋转珠并将抗体偶联的珠存储在60μlPBS(33%珠)中,加入0.05%NaN 3。 将珠子在4℃保存几个月。

  2. 细胞裂解
    1. 在7vol裂解缓冲液中裂解细胞
    2. 将裂解溶液在冰上30分钟至1小时
    3. 将裂解液在13,000rpm离心10分钟,4℃
    4. 转移上清液。 上清液可储存于-80℃备用。

  3. 共免疫沉淀
    1. 孵育3微升抗体偶联珠在250微升细胞裂解上清液,在4℃下旋转过夜
    2. 用含有500mM KCl和0.5%NP-40的20体积裂解缓冲液洗涤珠子4次,并用裂解缓冲液洗涤一次。
      注意:对于洗涤珠,我们通常在珠子中加入缓冲液,混合几次,倒转珠子,除去上清液。
    3. 用1×SDS蛋白凝胶样品上样缓冲液洗脱蛋白,通过SDS-PAGE(5-15%)分离并通过western印迹分析。

食谱

  1. PBST缓冲区
    PBS加0.1%Tween 20或0.1%Triton X-100
  2. 裂解缓冲液
    50mM Hepes(pH 7.4)
    200 mM KCl
    0.3%NP-40
    10%甘油 1 mM EGTA
    1mM MgCl 2
    0.5 mM DTT
    0.5μM微囊藻毒素
    10μgml -1 每种亮抑酶肽,胃蛋白酶抑制剂和抑糜蛋白酶
  3. 1x SDS蛋白凝胶样品上样缓冲液
    50mM Tris-HCl(pH6.8)
    2%SDS
    10%甘油 1%β-巯基乙醇 12.5mM EDTA
    0.02%溴酚蓝

致谢

该方案从在Guowei Fang博士(Stanford University,Stanford,CA,USA)的生物学实验室开发的免疫沉淀方案进行了修改。 这项工作得到了生物医学研究(G.F.)的Burroughs-Wellcome职业奖和国立卫生研究院(GM062852至G.F.)的资助。

参考文献

  1. Zhu,H.,Coppinger,J.A.,Jang,C.Y.,Yates,J.R.,3rdand Fang,G。(2008)。 FAM29A通过募集NEDD1-γ-微管蛋白复合物到有丝分裂纺锤体来促进微管扩增。 a> J Cell Biol 183(5):835-848。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Zhu, H. (2012). Immunoprecipitation for Cell Culture. Bio-protocol 2(3): e72. DOI: 10.21769/BioProtoc.72.
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