Research method of protein-protein interaction: co-immunoprecipitation
1. Wash appropriate cells on 30 10 cm plates with phosphate buffer. Scrape the cells from each plate into 1 ml of ice-cold EBC lysis buffer.
2. Transfer each ml of cell suspension to a microcentrifuge tube, and centrifuge at a maximum speed of 15 minutes at 4 ° C on a microcentrifuge.
3. Collect the supernatant (approximately 30 ml) and add 30 μg of appropriate antibody and shake the immunoprecipitate at 4 ° C for 1 h.
4. Add 0.9 ml of protein A-Sepharose suspension and shake the immunoprecipitate at 4 ° C for 30 min.
5. Wash the protein A-Sepharose mixture with NETN containing 900 mmol / L NaCl and repeat the washing 5 more times. Finally, wash once with NETN.
6. Aspirate the liquid portion of the mixture. Add 800 μl of 1 × SDS gel loading buffer to the beads and boil for 4 min.
7. The sample was added to a large-well discontinuous SDS-PAGE gradient gel and electrophoresed at a constant current of 10 mA overnight.
8. Observe the protein bands by Coomassie blue staining.
9. Cut the target band from the gel, place it in a microcentrifuge tube, and wash it twice with 1 ml of 50% acetonitrile for 3 min each time.
10. Digest the protein in the gel with trypsin, and then elute the peptide electrically.
11. Separation of peptides by narrow pore high performance liquid chromatography. Automatic Edman degradation sequencing of collected peptides on ABI 477A or 494A machines
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