免疫共沉淀实验检测酵母蛋白的相互作用

Olesya O. Panasenko

doi:10.21769/BioProtoc.250

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Peer-reviewed

Co-immunoprecipitation in Yeast

免疫共沉淀实验检测酵母蛋白的相互作用

Olesya O. Panasenko email

发布: 2012年08月20日第2卷第16期 DOI: 10.21769/BioProtoc.250 浏览次数: 23742

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参见作者原研究论文

The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

Feb 2012

Special Issue on Protocols for Organoid Research

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

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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

Glass beads for cells breaking 0.5 mm (Bio Spec Products, catalog number: 110/9105 )
Cycloheximide (CHX) (Sigma-Aldrich, catalog number: C7698 ) solution 100 mg/ml prepared on ethanol.
Bradford reactive (Bio-Rad Laboratories, catalog number: 500-0006 ).
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 )
Magnet, in case of using magnetic beads (Life Technologies, Invitrogen™, catalog number: 123.21D )
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.
Protease inhibitor cocktail (F. Hoffmann-La Roche, catalog number: 13560400 )
Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7625 ) 100 mM solution prepared on isopropanol
Laemmli sample buffer
Lysis buffer for protein complexes IP (see Recipes)
Lysis buffer for ribosome IP (see Recipes)