免疫共沉淀(Co-Immunoprecipitation)原理及实验方法-英文.docVIP

Principle: Co-Immunoprecipitation (Co-IP) was developed from the immunoprecipitation technique with which Co-IP shares the fundamental principle of the specific antigen-antiody reaction. Co-IP helps determine whether two proteins interact or not in physiological conditions in?vitro. Graphically, the Co-IP principle is as described in the right hand side picture. The known protein (antigen) is termed the bait protein, and the protein it interacts with is called the prey protein. The standard Co-IP protocol is the same as that described for IP, and actually any system designed for IP should also work for Co-IP. After that cells are completely lysed under non-denaturing conditions, proteins that bound together are kept. Therefore if you use anti-X to precipitate protein X through Co-IP, then you can get other proteins that interact with protein X in?situ. Co-IP is applied to test whether two known proteins bind each other in cells, or to find a new protein that interacts with a known protein. Advantages: Proteins that interact in a typical Co-IP are post-translationally modified and conformationally natural. In Co-IP proteins interact in a non-denaturing condition which is almost physiological. Disadvantages: The signals of low-affinity of protein interactions might not be detected. There might be a third protein in certain protein-protein interaction. To choose an appropriate antibody, the target protein needs to be properly predicted. Or there would not be a positive result in Co-IP. Reagents and buffers: PBS RIPA (RadioImmunoPrecipitation Assay) Lysis buffer: ? Tris-HCl: 50 mM, pH 7.4 ? Nonidet P-40 (NP-40): 1% ? Deoxycholate Na：0.25% ? NaCl: 120 mM ? EDTA: 1 mM ? *PMSF: 1 mM ? *Leupeptin 1 μg/ml ? *Aprotinin 1 μg/ml ? *Pepstatin1 μg/ml ? *Na3VO4: 1 mM ? *NaF: 1 mM Note:?Ingredients labeled with * should be added right before each use. PMSF degrades to a half after 30min in water. Washing buffer : lysis buffer:5M NaCl =100:1 (ensure NaCl not exceed 1M) protein A/G-a