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Advantages and disadvantages of co-immunoprecipitation
Advantages: 1. The protein interaction is carried out in a natural state, which can avoid human influence.
2. The interacting protein complex in its natural state can be isolated.
3. The interacting proteins are all modified after translation and are in their natural state.
Disadvantages: 1. Low affinity and instant protein-protein interaction may not be detected;
2. It is necessary to predict what the target protein is before the experiment in order to select the final antibody to be detected, so if the prediction is incorrect, the experiment will not get results, and the method itself is risky;
3. The combination of the two proteins may not be a direct combination, but a third party may act as a bridge in the middle.
The following is a step-by-step method for determining the binding protein by co-immunoprecipitation that Hengyuan Biotech has compiled for friends:
1. Wash 30 suitable cells on 10 cm culture plates with phosphate buffer. Scrape the cells from each plate into 1 ml ice-cold EBC lysis buffer;
2. Transfer each ml of cell suspension to a microcentrifuge tube and centrifuge at a maximum speed of 4 ° C for 15 ° C on a microcentrifuge;
3. Collect the supernatant (about 30 ml) and add 30 μg of the appropriate antibody, 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 washing 5 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 ball and boil for 4 min;
7. Add the sample to a large-pore discontinuous SDS-PAGE gradient gel and run it overnight at a constant current of 10 mA;
8. Observe the protein swimming band by Coomassie brilliant blue staining;
9. Cut the target tape from the gel, place it in a microcentrifuge tube, and wash it twice with 1 ml 50% acetonitrile for 3 min each time;
10. Digest the protein in the gel with trypsin, then elute the peptide electrically;
11. Separation of peptides by narrow pore high performance liquid chromatography. The collected peptides were subjected to automated Edman degradation sequencing on ABI 477A or 494A machines.
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