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Immunoprecipitation technology service

Shanghai Jinma Experimental Equipment provides accurate immunoprecipitation technology operation steps, welcome to read and appreciate!

Immunoprecipitation is mainly used for the qualitative detection of antigens or antibodies. The principle refers to the phenomenon of visible precipitate formed by the soluble antigen and the corresponding antibody in the presence of an electrolyte in an appropriate ratio. The precipitation experiments designed according to this phenomenon mainly include flocculation precipitation test, cyclic precipitation test and precipitation test in gel. The precipitation test in the gel can be further divided into two types according to the experimental methods used in the immunodiffusion experiment and the immunoelectrophoresis technique.

Co-Immunoprecipitation is a classical 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 basic principle is that an antibody is added to the cell lysate to form a specific immune complex with the antigen, and the immune complex is collected after elution, and then subjected to SDS-PAGE and Western blotting analysis.

Immunoprecipitation procedure (taking the Protein A Agarose method as an example):
1. Preparation of protein samples
• For adherent cells in a 10 cm cell culture dish, aspirate the cell culture medium, wash once with PBS, and then lyse the cells by adding 500 μl to 2 ml of cell lysate.
• Lysis is performed on the proportion of lysate used in tissue samples with reference to adherent cells.
• For suspension cells, the cells were collected by centrifugation, washed once with PBS, and then lysed by reference to the lysis method of adherent cells.

2. Remove non-specific binding
Take 200 microliters to 1 milliliter of protein sample, the amount of protein is about 200 micrograms to 1 milligram, add about 1 microgram and the same common IgG used in immunoprecipitation and 20 microliters of fully resuspended Protein A Agarose. Shake slowly for 4 minutes to 2 hours at 4 °C.
• Centrifuge at 2500 rpm (about 1000 g) for 5 minutes, and take the supernatant for subsequent immunoprecipitation.

3. Immunoprecipitation
• Add 0.2-2 micrograms of primary antibody for immunoprecipitation and slowly shake overnight at 4 °C.
• Add 20 μl of fully resuspended Protein A Agarose and shake slowly for 1-3 hours at 4 °C.
• Centrifuge at 2500 rpm (approximately 1000 g) for 5 minutes, or instantaneously centrifuge at high speed, carefully aspirate the supernatant, taking care to leave a small amount of supernatant and not to aspirate Protein A Agarose.
• Wash the pellet 5 times with the lysate or PBS when preparing the protein sample, and the amount of the lysate or PBS is 0.5-1 ml each time. The centrifugation conditions during washing and the requirements for aspirating the supernatant are the same as those described above.
After the last wash, the supernatant was removed, and 20-40 μl of 1X SDS-PAGE electrophoresis loading buffer Vortex was added to resuspend the pellet, and the sample was centrifuged to the bottom of the tube by instantaneous high-speed centrifugation.
● 100 ° C or boiling water bath for 3-5 minutes, take part or all of the sample for SDS-PAGE electrophoresis, temporarily unused samples can be stored at -20 ° C.

Immunoprecipitation:
Reference is made to the method of immunoprecipitation, but co-IP co-precipitation (co-IP) usually requires the use of fresh protein samples that have not been frozen. Ordinary immunoprecipitations may use frozen protein samples, but fresh protein samples are preferred.