Note: This protocol provides a general method suitable for the immunoprecipitation of radio-labelled antigens. Please note that the choice of radio-labelling procedure may be important in the design of an immunoprecipitation experiment. For the labelling of cell surface antigens lactoperoxidase catalysed iodination with 125l is recommended, whilst 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 be used throughout

Appropriate safety precautions should be taken when working with any radioactive isotopes.

1. If using radio-labelled cells lyse 2 x 10⁷ cells in 0.5ml of cold solubilizing solution. Incubate for 20 minutes on ice.

2. Centrifuge at 1000g for 10 minutes to remove insoluble debris.

3. Add lysate to 200ml of Protein G-Sepharose beads, and incubate overnight at 4^oC with tumbling to remove any material that may bind non-specifically to the beads.

4. Simultaneously add 200ml of the monoclonal antibody of interest (at approximately 10-50mg/ml) to 100ml of Protein-G Sepharose beads, and incubate with tumbling overnight at 4^oC.

5. Remove beads from cell lysate by centrifugation (11,000g. 5 minutes).

6. Wash monoclonal antibody loaded beads twice in phosphate buffered saline, and remove supernatant following centrifugation.

7. Add cell lysate to monoclonal antibody loaded beads, and incubate at 4^oC for at least 90 minutes with tumbling.

8. Wash beads 3 times with cold solubilizing solution, harvesting each time by centrifugation.

9. Analyse immunoprecipitate by resuspending beads in SDS-PAGE sample buffer, and running on a suitable SDS-PAGE gel. The gel should be dried and analysed by autoradiography after a suitable exposure period.

Solubilizing solution

mg/100ml PBS

EDTA 37.0

EGTA 38.0

Iodoacetamide 93.0

Phenylmethylsulfonylfluoride 17.4

Soybean trypsin inhibitor 2.0

Aprotinin (20KIU/ml) 100ml