Metabolic labeling Cells
Dominik Haudenschild after protocol by Urs Hofer, June 1994 CBRC
Procedure
1. Plate cells at a density of 10
6 cells per 6 cm dish in DMEM/10% FCS (about 60% to 80% confluent)2. Incubate cells overnight, allow to attach to plastic dish
3. Remove media, and wash cells with 4ml DMEM without cysteine/methionine
4. Add 2ml labeling solution per 6cm dish
5. Incubate cells for about 15 to 20 hours at 37°C. To entrap volatile decomposition products of the
35S amino acids, place culture dish into a half open plastic box containing an open dish of activated charcoal. Charcoal will be radioactive.6. Collect conditioned medium and cells and perform the desired experiment
Solutions
1. Standard DMEM with 10% fetal calf serum or newborn calf serum
2. Labeling solution:
mix: DMEM without cysteine and methionine 1.6ml
Tran