Immunoprecipitation Protocol for Labeled Cells.

- from An Zhou, Johns Hopkins, 1995. Click here for full protocol. 

1. To your sample, add Super E, protease inhibitors, 1 mM cold Met, antibody.

TMT extract or medium - dilute with Super E to convenient volume; add protease inhibitors, cold Met and antibody.

Some antibodies work only after the antigen has been denatured (e.g. Ab 877, Ab 471): to 50 ul of sample in TMT, add 5 µl 10% SDS and incubate at 100 ^oC for 5 min; allow to cool to room temperature for 5 min; add IN THIS ORDER: 25 µl 15% NP-40, 180 µl Super E, 3 µl PMSF, 3 µl inhibitor mix, 10 µl antibody. Super E: 50 mm phosphate, 1% Triton.