搜索

Co-immunoprecipitation in Yeast
免疫共沉淀实验检测酵母蛋白的相互作用   

下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

Abstract

This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically binds to that particular protein (antigene protein). The partner proteins that are bound to a specific target protein can be co-immunoprecipitated together with an antigen. These protein complexes can then be analyzed to identify new binding partners, binding affinities, the kinetics of binding and the function of the target protein. Here I describe the protocols that allow to immunoprecipitate different protein complexes, for example NAC complex (Panasenko et al., 2009), Ccr4-Not complex (Panasenko and Collort, 2011), ribosomes (Panasenko and Collort, 2012) and investigate their partners. For each CoIP I used the different lysis buffer, as indicated below in recipes.

Keywords: Immunoprecipitation(免疫共沉淀), Protein interaction(蛋白质相互作用), Affinity interaction(亲和作用), Antibody(抗体), Yeast(酵母)

Materials and Reagents

  1. Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
  2. Cycloheximide (CHX) (Sigma-Aldrich, catalog number:  C7698 ) solution 100 mg/ml prepared on ethanol.
  3. Bradford reactive (Bio-Rad Laboratories, catalog number:  500-0006 ).
  4. Different types of the beads can be used depending on the particular antigene protein and antibodies.
    1. Protein G magnetic Dynabeads (Life Technologies, Invitrogen™, catalog number:  100.04D ) or Protein A magnetic Dynabeads (Life Technologies, Invitrogen™, catalog number:  100.02D )
    2. Protein G Sepharose (Amersham biosciences, catalog number: 17-0618-01 ) or Protein A Sepharose (Amersham biosciences, catalog number: 17-0780-01 )
  5. Magnet, in case of using magnetic beads (Life Technologies, Invitrogen™, catalog number:  123.21D )
  6. Antibodies
    For example Peroxidise-anti-peroxidase soluble complex (PAP-antibodies) were bought from Sigma-Aldrich (catalog number: P1291 ). Antibodies against HA (HA.11) (Clone 16B12, catalog number: MMS-101R ) and Myc [c-myc (9E10), catalog number: MMS-150R ] were bought from Covance.
  7. Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number:  13560400 )
  8. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number:  P7625 ) 100 mM solution prepared on isopropanol
  9. Laemmli sample buffer
  10. Lysis buffer for protein complexes IP (see Recipes)
  11. Lysis buffer for ribosome IP (see Recipes)

Equipment

  1. Table Centrifuges
  2. Glass bead beater Genie Disruptor (Scientific Industries, catalog number: SI-DD38 )

Procedure

  1. Nascent associated complex (NAC) and Ccr4-Not complex IP
    1. Yeast cultures were grown on YPD or selective media. In the morning dilute night culture till OD600 = 0.15. Grow 100 ml of culture at 30 °C until OD600 = 0.6-0.8.
    2. Centrifuge the cells for 5 min at 4,000 x g. Wash the cells with 1 ml of sterile H2O and transfer to the eppendorf tube. Cells can be stored at -20 °C.
    3. Work on ice! Resuspend the pellet in 0.5 ml of lysis buffer, add 0.5 ml of glass beads and disrupt 15 min on the glass bead beater at 4 °C.
    4. Transfer the liquid phase into the new tube. Wash the beads with 0.5 ml of lysis buffer. Combine all liquid phases.
    5. To avoid the contamination with cells derby preclean the total extracts. For this transfer the liquid phase into the new tube. Centrifuge 1 min 16,000 x g at 4 °C.
    6. Transfer the supernatant into the new eppendorf tube and centrifuge 20 min 16,000 x g at 4 °C.
    7. Measure the total protein concentrations in the supernatants [= TE (Total Extracts)]. For this dilute the total extracts 1: 20 and load 5 and 10 μl to the Bradford sample (0.8 ml of water and 0.2 ml of Bradford reactive).
    8. Use Protein-A or Protein-G beads 20 μl of 100% beads per reaction. Wash the beads for 20 sec 2 times with 1 ml of water and 1 time with 1 ml of lysis buffer, after each washing spin the beads at 800 x g for 1 min. Resuspend till 20% in lysis buffer. Take 100 μl of this suspension per reaction.
    9. Mix 0.4 ml of the total extracts containing 2 mg of total protein (5 mg/ml) with 100 μl of beads suspension and 1-2 μl of antibodies.
    10. Incubate from 4 h till over night at 4 °C with mild rotation.
    11. Wash the beads for 20 sec with 1 ml of lysis buffer 3 times, after each washing spin the beads at 800 x g for 1 min.
    12. Add 50 μl of Laemmli sample buffer 2x to the beads and incubate 10 min at 65 °C.
    13. Analyze the CoIP by SDS-PAGE and western blot. Load the total extracts (30 μg) and immunoprecipitated fraction (20 μl) on the gel.
      Notes: For the control there are 2 possibilities. 
      1. Use the total extracts from the strains lacking the immunoprecipitated antigen.
      2. Use the same total extract that you use for IP but do not add the antibodies.

  2. Ribosome IP
    On the same mRNA to form polysomes. It is possible to immunoprecipitate separate ribosome particles (40S or 60S) or polysomes. To disrupt the polysomes, 25 mM of EDTA is added to the buffers. To maintain the polysomes, 0.1 mg/ml of cycloheximide (CHX) is added to the culture before collection and to all the solution.
    1. Yeast cultures were grown on YPD or selective media. In the morning dilute night culture till OD600 = 0.15. Grow 100 ml of culture at 30 °C until OD600 = 0.6-0.8.
    2. To keep the polysomes add cycloheximide (CHX) till final concentration 0.1 mg/ml and incubate 10 min on ice. To disrupt the polysomes avoid this step.
    3. To keep the polysomes spin the cells for 5 min at 4,000 x g and wash with 50 ml of cold water containing 0.1 mg/ml of CHX. To disrupt the polysomes wash with 50 ml of cold water without CHX.
    4. Resuspend the pellets in 1 ml of lysis buffer containing 0.1 mg/ml of CHX (in case of polysomes), spin. Cells can be frozen and stored at -20 °C.
    5. Transfer the liquid phase into the new tube. Wash the beads with 0.5 ml of lysis buffer. Combine all liquid phases.
    6. To avoid the contamination with cells derby preclean the total extracts. For this transfer the liquid phase into the new tube. Centrifuge 1 min 16,000 x g at 4 °C.
    7. Transfer the supernatant into the new eppendorf tube and centrifuge 20 min 16,000 x g at 4 °C.
    8. Measure the total protein concentrations in the supernatants [= TE (total extracts)]. For this dilute the total extracts 1: 20 and load 5 and 10 μl to the Bradford sample (0.8 ml of water and 0.2 ml of Bradford reactive).
    9. Use Protein-A or Protein-G beads 20 μl of 100% beads per reaction. Wash the beads for 20 sec 2 times with 1 ml of water and 1 time with 1 ml of lysis buffer, after each washing spin the beads at 800 x g for 1 min. Take 100 μl of this suspension per reaction.
    10. Mix 0.4 ml of the total extracts containing 2 mg of total protein (5 mg/ml) with 100 μl of beads suspension and 1-2 μl of antibodies.
    11. Incubate from 2-4 h at 4 °C with mild rotation.
    12. Wash the beads for 20 sec with 1 ml of lysis buffer, containing 0.5 % of Triton X-100, 3 times. After each washing spin the beads at 800 x g for 1 min.
    13. Add 50 μl of Laemmli sample buffer 2x to the beads and incubate 10 min at 65 °C.
    14. Analyze the CoIP by SDS-PAGE and western blot. Load the total extracts (30 μg) and immunoprecipitated fraction (20 μl) on the gel.
      Notes: For the control there are 2 possibilities.
      1. Use the total extracts from the strains lacking the immunoprecipitated antigen.
      2. Use the same total extract that you use for IP but do not add the antibodies.

Recipes

  1. Lysis buffer for protein complexes (for example NAC or Ccr4-Not complex) IP (Panasenko et al., 2009; Panasenko and Collort, 2011)
    Usually we use 2 types of the buffers. In both of them protein complexes can be efficiently immunoprecipitated.
    Lysis buffer 1 (Panasenko et al., 2009)
    40 mM HEPES-KOH (pH 7.5)
    100 mM KCl
    150 mM K-acetate
    1 mM EDTA (pH 8.0)
    20% glycerol
    Lysis buffer 2 (Panasenko and Collort, 2011)
    50 mM Tris-HCl (pH 8.0)
    100 mM NaCl
    1 mM EDTA
    5 mM MgCl2
    1 mM DTT
    10% glycerol
    0.5 mM PMSF
  2. Lysis buffer for ribosome IP (Panasenko and Collort, 2012)
    10 mM Tris-HCl (pH 7.5)
    100 mM NaCl
    30 mM MgCl2
    0.1 % Triton X-100
    1 mM PMSF
    Note: All solutions contain 0.1 mg/ml of CHX, in case of keeping polysomes, or 25 mM of EDTA, in case of polysomes disruption.

Acknowledgments

This work was supported by grants from Ernst and Lucie Schmidheiny Foundation and Pierre Mercier Foundation awarded to O.O.P. and grants 31003A-120419 and 31003A_135794 of the Swiss National Science Foundation as well as a grant from the Novartis Foundation awarded to M.A.C.

References

  1. Panasenko, O. O. and Collart, M. A. (2011). Not4 E3 ligase contributes to proteasome assembly and functional integrity in part through Ecm29. Mol Cell Biol 31(8): 1610-1623.
  2. Panasenko, O. O. and Collart, M. A. (2012). Presence of Not5 and ubiquitinated Rps7A in polysome fractions depends upon the Not4 E3 ligase. Mol Microbiol 83(3): 640-653.
  3. Panasenko, O. O., David, F. P. and Collart, M. A. (2009). Ribosome association and stability of the nascent polypeptide-associated complex is dependent upon its own ubiquitination. Genetics 181(2): 447-460.

简介

该协议描述了通过共免疫沉淀(CoIP)对面包酵母中蛋白质 - 蛋白质相互作用的研究。 CoIP是鉴定细胞中生理相关的蛋白质 - 蛋白质相互作用的技术。 使用特异性结合该特定蛋白(抗原蛋白)的抗体可以从溶液中分离出感兴趣的蛋白。 与特定靶蛋白结合的伴侣蛋白可以与抗原一起免疫共沉淀。 然后可以分析这些蛋白质复合物以鉴定新的结合配偶体,结合亲和力,结合的动力学和靶蛋白的功能。 这里我描述允许免疫沉淀不同蛋白复合物的方案,例如NAC复合物(Panasenko等人,2009),Ccr4-Not复合物(Panasenko和Collort,2011),核糖体(Panasenko和Collort ,2012),并调查他们的合作伙伴。 对于每个CoIP,我使用不同的裂解缓冲液,如下文配方中所示。

关键字:免疫共沉淀, 蛋白质相互作用, 亲和作用, 抗体, 酵母

材料和试剂

  1. 用于破碎细胞0.5mm的玻璃珠(Bio Spec Products,目录号:110/9105)
  2. 用乙醇制备的环己酰亚胺(CHX)(Sigma-Aldrich,目录号:C7698)溶液100mg/ml。
  3. Bradford反应性(Bio-Rad Laboratories,目录号:500-0006)。
  4. 可以使用不同类型的珠,这取决于特定的抗原基因蛋白和抗体。
    1. 蛋白G磁性Dynabeads(Life Technologies,Invitrogen TM,目录号:100.04D)或Protein A磁性Dynabeads(Life Technologies,Invitrogen TM,目录号:100.02D)
    2. 蛋白G Sepharose(Amersham biosciences,目录号:17-0618-01)或Protein A Sepharose(Amersham biosciences,目录号:17-0780-01)
  5. 磁体,在使用磁珠的情况下(Life Technologies,Invitrogen TM,目录号:123.21D)
  6. 抗体
    例如,过氧化物 - 过氧化物酶可溶性复合物(PAP-抗体)购自Sigma-Aldrich(目录号:P1291)。 针对HA(HA.11)(克隆16B12,目录号:MMS-101R)和Myc [c-myc(9E10),目录号:MMS-150R]的抗体购自Covance。
  7. 蛋白酶抑制剂混合物(F.Hoffmann-La Roche,目录号:13560400)
  8. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7625)在异丙醇上制备的100mM溶液
  9. Laemmli样品缓冲液
  10. 蛋白质复合物的溶解缓冲液IP(参见配方)
  11. 核糖体IP的裂解缓冲液(参见配方)

设备

  1. 台离心机
  2. 玻璃珠打浆机Genie Disruptor(Scientific Industries,目录号:SI-DD38)

程序

  1. 初始相关复合物(NAC)和Ccr4-非复杂IP
    1. 酵母培养物在YPD或选择性培养基上生长。 在早晨稀释夜培养直至OD <600> = 0.15。 在30℃下生长100ml培养物,直到OD 600 = 0.6-0.8。
    2. 在4,000×g离心细胞5分钟。 用1ml无菌H 2 O洗涤细胞并转移到eppendorf管中。 细胞可以储存在-20°C
    3. 在冰上工作! 将沉淀重悬于0.5ml裂解缓冲液中,加入0.5ml玻璃珠,在4℃下在玻璃珠打浆机上破碎15分钟。
    4. 将液相转移到新管中。用0.5ml裂解缓冲液洗涤珠子。合并所有液相。
    5. 为了避免细胞污染德比预先清除总提取物。为了将液相转移到新管中。在4℃下离心1分钟16,000×g
    6. 将上清液转移到新的eppendorf管中,并在4℃下离心20分钟16,000×g。
    7. 测量上清液中的总蛋白浓度[= TE(总提取物)]。对于这种稀释,总提取物1:20,并加载5和10μl到Bradford样品(0.8ml水和0.2ml Bradford反应性)。
    8. 使用蛋白A或蛋白G珠20μl的100%珠每个反应。用1ml水洗涤珠子20秒2次,用1ml裂解缓冲液洗涤1次,每次洗涤后,将珠子以800×g离心1分钟。在裂解缓冲液中重悬至20%。取每个反应100微升的这个悬浮液。
    9. 将0.4ml含有2mg总蛋白(5mg/ml)的总提取物与100μl珠悬浮液和1-2μl抗体混合。
    10. 孵育4小时,直到过夜,在4°C轻度旋转。
    11. 用1ml裂解缓冲液洗涤珠子20秒,每次洗涤后将珠子以800×g离心1分钟。
    12. 向珠子中加入50μlLaemmli样品缓冲液2x,并在65℃下孵育10分钟
    13. 通过SDS-PAGE和western印迹分析CoIP。 将总提取物(30μg)和免疫沉淀的部分(20μl)装载到凝胶上 注意:对于控件,有2种可能性。
      1. 使用缺乏免疫沉淀抗原的菌株的总提取物。
      2. 使用与IP相同的总摘录,但不添加抗体。

  2. 核糖体IP
    在相同的mRNA上形成多核糖体。可以免疫沉淀分开的核糖体颗粒(40S或60S)或多核糖体。为了破坏多核糖体,将25mM EDTA加入到缓冲液中。为了保持多核糖体,在收集之前向培养物中加入0.1mg/ml的放线菌酮(CHX)并加入到所有溶液中。
    1. 酵母培养物在YPD或选择性培养基上生长。在早晨稀释夜培养直至OD <600> = 0.15。在30℃下生长100ml培养物,直到OD 600 = 0.6-0.8。
    2. 保持多核糖体添加放线菌酮(CHX)直至最终浓度为0.1mg/ml,并在冰上孵育10分钟。破坏多核糖体避免这一步。
    3. 保持多核糖体以4,000×g离心细胞5分钟,并用50ml含有0.1mg/ml CHX的冷水洗涤。为了破坏多核糖体用50ml冷水洗涤,不用CHX。
    4. 将沉淀重悬在1ml含有0.1mg/ml CHX(在多核糖体的情况下)的裂解缓冲液中,旋转。可以将细胞冷冻并储存在-20℃。
    5. 将液相转移到新管中。用0.5ml裂解缓冲液洗涤珠子。合并所有液相。
    6. 为了避免细胞污染德比预先清除总提取物。为了将液相转移到新管中。在4℃下离心1分钟16,000×g。
    7. 将上清液转移到新的eppendorf管中,并在4℃下离心20分钟16,000×g 。
    8. 测量上清液中的总蛋白浓度[= TE(总提取物)]。对于这种稀释,总提取物1:20,并加载5和10μl到Bradford样品(0.8ml水和0.2ml Bradford反应性)。
    9. 使用蛋白A或蛋白G珠20μl的100%珠每个反应。用1ml水洗涤珠子20秒2次,用1ml裂解缓冲液洗涤1次,每次洗涤后将珠子在800× g 1分钟。 每次反应取100μl此悬浮液。
    10. 将0.4ml含有2mg总蛋白(5mg/ml)的总提取物与100μl珠悬浮液和1-2μl抗体混合。
    11. 在4°C下孵育2-4小时,轻微旋转
    12. 用1ml含有0.5%Triton X-100的裂解缓冲液洗涤珠子20秒,3次。 每次洗涤后,将珠子以800×g离心1分钟
    13. 向珠子中加入50μlLaemmli样品缓冲液2x,并在65℃下孵育10分钟
    14. 通过SDS-PAGE和western印迹分析CoIP。 将总提取物(30μg)和免疫沉淀的部分(20μl)装载到凝胶上 注意:对于控件有2种可能性。
      1. 使用缺乏免疫沉淀抗原的菌株的总提取物。
      2. 使用与IP相同的总摘录,但不添加抗体

食谱

  1. 用于蛋白质复合物(例如NAC或Ccr4-Not复合物)的溶解缓冲液IP(Panasenko等人,2009; Panasenko和Collort,2011)
    通常我们使用2种类型的缓冲区。 在它们两者中,蛋白质复合物可以有效地免疫沉淀 裂解缓冲液1(Panasenko et al。,2009)
    40mM HEPES-KOH(pH7.5) 100 mM KCl
    150 mM K-acetate
    1mM EDTA(pH8.0) 20%甘油 裂解缓冲液2(Panasenko和Collort,2011)
    50mM Tris-HCl(pH8.0)
    100 mM NaCl
    1mM EDTA
    5mM MgCl 2/
    1 mM DTT
    10%甘油 0.5 mM PMSF
  2. 核糖体IP的裂解缓冲液(Panasenko和Collort,2012)
    10mM Tris-HCl(pH7.5) 100 mM NaCl
    30mM MgCl 2/v/v 0.1%Triton X-100 1mM PMSF
    注意:在多核糖体破坏的情况下,所有溶液含有0.1mg/ml的CHX,以防多核糖体或25mM的EDTA。

致谢

这项工作得到Ernst和Lucie Schmidheiny基金会和Pierre Mercier基金会授予O.O.P.的资助。 并授予瑞士国家科学基金会的31003A-120419和31003A_135794以及授予M.A.C.的诺华基金会赠款。

参考文献

  1. Panasenko,O.O.和Collart,M.A。(2011)。 Not4 E3连接酶通过Ecm29部分有助于蛋白酶体装配和功能完整性。 Mol Cell Biol 31(8):1610-1623。
  2. Panasenko,O.O.和Collart,M.A。(2012)。 Not5和泛素化Rps7A在多核糖体级分中的存在取决于Not4 E3连接酶。 em> Mol Microbiol 83(3):640-653。
  3. Panasenko,O.O.,David,F.P.and Collart,M.A。(2009)。 新生多肽相关复合物的核糖体结合和稳定性取决于其自身的泛素化。 181(2):447-460
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Panasenko, O. O. (2012). Co-immunoprecipitation in Yeast. Bio-protocol 2(16): e250. DOI: 10.21769/BioProtoc.250.
提问与回复

(提问前,请先登录)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。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。