Selenomethionine Labeling of Secreted Proteins in 293 Cells

Prepare media for labeling:

1. Add 30 mg/L L-cystine-2HCl to DME-HG lacking Met. & Cys. (Gibco 21013-024).

2. Add 60 mg/L L-selenomethionine.

3. Let sit overnight at room temperature to allow the cys. & se-met. to go into solution.

4. Filter sterilize and add Pen./Strep. and L-glutamine to 2mM (Do not add glutamine before this step!)

Label the cells:

1. To cells that are ~90% confluent, aspirate away media, and wash with either PBS or media w/o Met. and Cys. (preferred).

2. Add Se-Met media to the bottles and let incubate for 12 hours.

3. Remove the media and feed with fresh media. Throw this media out. This step serves to deplete intracellular methionine pools.

4. Continue to incubate for 48-72 hours.

**Cells labeled in this way have ~92-93% incorporation of seleno-methionine.