Metabolic labeling of proteins

1. Grow cells in 6-well plate. If cells are transfected, label cells 24-48 hours after transfection.

2. Rinse cells twice with methionine and cystine free growth media.

3. (optional) Add 1.5 ml methionine and cystine free growth media supplemented with 10% dialyzed FCS. Culture 4-16 hours.

4. Add 20-30 ml of Expre35S35S ( 35S-Express )protein labeling mix (NEN). Label cells at 37 C for 1-2 hours.

5. Add 1.5 ml normal media with methionine, cystine, and 10% serum. Chase 4 hour to over night at 37 C.

6. Remove media, and wash twice with PBS, and harvest cells.