Protocol for Immunoprecipiation of Cultured Cells

1. Incubate cells at ~80% confluent in medium with 10% FBS lacking the labeled amino acid for approximately 1 hour at 37^oC
2. Remove media and replace with fresh media lacking the labeled amino acid with 10% FBS
3. Add the radioactively labelled amino acid (approximately 100uCi/ml)
4. Incubate at 37^oC for 1hour to overnight. This step may require some optimization. While most proteins will be labeled after 1 hour, others may take overnight incubation
5. Carefully remove radioactive media and dispose
6. Add 6ml PBSTDS (4^oC) to cells or cell pellet and incubate for 10 minutes
7. Mechanically disrupt cells by repeatedly drawing mixture through a 21 gauge needle
8. Wash incubation plate with an additional 4ml of PBSTDS (4^oC)
9. Centrifuge at 2,000rpm for 15 minutes in a refrigerated centrifuge
10. Discard pellet
11. Centrifuge supernatant at 40,000rpm for 20 minutes in a refrigerated centrifuge
12. Discard pellet
13. Aliquot approximately 1ml of supernatnat to 1.5ml eppendorf tubes
14. Add approximately 1ug antibody to target protein and incubate for 1-2 hours at 4^oC. The concentration requires optimization
15. Add conjugate antibody. Several options are available here and I have commonly used agarose conjugates
16. Incubate at 4^oC on rocker platform for 12-24 hours (requires optimization - may need less time)
17. Collect immunoprecipiatated protein by centrifugation at 2500rpm for 15 minutes at 4^oC
18. Wash pellet 3-4 times with 1ml PBSTDS
19. Resuspend in 40ul gel running buffer
20. Boil samples for 3 minutes to denature prior to SDS-PAGE analysis

Note: Please store, incubate, and dispose of radioactive materials responsibly!