Co-immunoprecipitation experiment method Co-immunoprecipitation is a classic method for studying protein interactions based on the specific interaction between antibodies and antigens. It is an effective method to determine the physiological interaction of two proteins in intact cells. The principle is that when cells are lysed under non-denaturing conditions, many protein-protein interactions that exist within intact cells are preserved. If x is immunoprecipitated with antibodies to protein x, protein y bound to x in the body can also be precipitated. This method is often used to determine whether two target proteins are bound in the body; it can also be used to determine a new partner for a specific protein. Its advantages are: (1) The interacting proteins are all post-translationally modified and in a natural state; (2) The protein interactions are performed in a natural state, which can avoid human influence; (3) It can be isolated Interacting protein complexes in their natural state. The disadvantages are: (1) low affinity and instant protein-protein interaction may not be detected; (2) the combination of two proteins may not be a direct combination, but a third party may act as a bridge in the middle; (3) must Predict what the target protein is before the experiment to select the final antibody to be tested, so if the prediction is not correct, the experiment will not get results, and the method itself is risky. The experimental process is: (1) Cells can be harvested 24-48 h after transfection, add an appropriate amount of cell lysis buffer (containing protease inhibitors), lyse on ice for 30 min, cell lysate at 4 ° C, and centrifuge at maximum speed for 30 min to take the supernatant; (2) Take a small amount of lysate for western blot analysis, add 1μg of the corresponding antibody to the remaining lysate and add it to the cell lysate. Incubate overnight at 4 ° C with gentle shaking; (3) Take 10μl protein a agarose beads, wash with appropriate amount of lysis buffer 3 times, centrifuge at 3,000 rpm for 3 min each time; (4) Add the pretreated 10μl protein a agarose beads to the cell lysate incubated with the antibody overnight and incubate slowly at 4 ° C for 2-4h, to couple the antibody with the protein a agarose beads; (5) After the immunoprecipitation reaction, centrifuge at 3,000 rpm for 3 min at 4 ° C, centrifuge the agarose beads to the bottom of the tube; carefully aspirate the supernatant, and wash the agarose beads 3-4 times with 1 ml of lysis buffer; Finally, add 15μl of 2 × sds loading buffer and cook in boiling water for 5 minutes; (6) Sds-page, western blotting or mass spectrometer analysis. Issues to note: (1) Mild lysis conditions are used for cell lysis, which cannot destroy all protein-protein interactions present in the cell, and non-ionic denaturants (np40 or triton x-100) are mostly used. The lysis conditions for each cell are different and determined empirically. High-density denaturants (0.2% sds) cannot be used. Various enzyme inhibitors, such as commercial cocktailers, should be added to the cell lysate. (2) Use clear antibodies, you can use several antibodies together (3) Use control antibody: Monoclonal antibody: normal mouse igg or another type of monoclonal antibody Rabbit polyclonal antibody: normal rabbit igg To ensure the authenticity of the experimental results in the immunoprecipitation experiment, the following points should be noted: (1) Ensure that the co-precipitated protein is precipitated by the added antibody, rather than foreign non-specific protein. The use of monoclonal antibody helps to avoid the occurrence of contamination; (2) It is necessary to ensure the specificity of the antibody, that is, the addition of the antibody to the cell lysate that does not express the antigen will not cause co-precipitation; (3) It is determined that the interaction between proteins occurs in cells, rather than due to cell lysis. This requires the positioning of proteins to determine. Bathroom Organizer,Bathroom Storage Rack ,Bathroom Towel Shelf,Towel Storage Rack Jiangmen Sunbond Houseware Manufacturing CO.,LTD , https://www.sunbondhw.com
Co-immunoprecipitation experiment method