Protocol: Immunoprecipitation

This protocol provides a general method suitable for the immunoprecipitation of radiolabeled antigens. Please note that the choice of radio-labeling procedure may be important in the design of an immunoprecipitation experiment. For the labeling of cell surface antigens, lactoperoxidase catalyzed iodination with 125l is recommended, while for intracellular antigens in cultured cells, 35S-methionine incorporation would be more suitable (see Practical Immunology, Hudson, C. and Hay, F.C. Blackwell Scientific Publications).

We recommend that suitable controls are used throughout. Appropriate safety precautions should be taken when working with any radioactive isotopes.

Reagents

1. Solubilizing solution
To 100 ml of Phosphate Buffered Saline (PBS) add:

EDTA, 37.0 mg
EGTA, 38.0 mg
Iodoacetamide, 93.0 mg
Phenylmethylsulfonylfluoride, 17.4 mg
Soybean trypsin inhibitor, 2.0 mg
Aprotinin (20 KIU/ml), 100 μl

Method

1. If using radio-labeled cells, lyse 2 x 107 cells in 0.5 ml of cold solubilizing solution. Incubate for 20 minutes on ice.
2. Centrifuge at 1000 g for 10 minutes to remove insoluble debris.
3. Add lysate to 200 μl of Protein G-Sepharose beads, and incubate overnight at 4°C with tumbling. This precautionary step will remove any material from the lysate that may bind non-specifically to the beads.
4. At the same time in a separate tube, add 200 μl of the monoclonal antibody of interest (at approximately 10-50 μg/ml) to 100 μl of Protein-G Sepharose beads. Incubate with tumbling overnight at 4°C.
5. Remove beads from cell lysate suspension by centrifugation (11,000 g for 5 minutes).
6. Wash monoclonal antibody-loaded beads twice in PBS. Discard the supernatant following centrifugation.
7. Add the cell lysate to the monoclonal antibody-loaded beads and incubate at 4°C for at least 90 minutes with tumbling.
8. Wash the beads 3 times with cold solubilizing solution, harvesting them each time by centrifugation.
9. Analyze immunoprecipitate by eluting beads with a small volume of SDS-PAGE loading buffer. Run the eluate on a suitable SDS-PAGE gel. The gel should be dried and analyzed by autoradiography following a suitable exposure period.