Preparation of SILAC Media

Reagents

Arg‐ and Lys‐free Media: DMEM, RPMI‐1640, Phenol red‐free MEM and 1:1 DMEM:F12 are available from Pierce. SILAC media is also available from Sigma‐Aldrich and Invitrogen. Several companies also offer custom media services.

Fetal Bovine Serum: FBS that has been dialyzed using a 10 kDa cut‐off membrane (Sigma‐Aldrich: F0392) is available in 100and 500 ml bottles from the Duke CCF (http://www.cancer.duke.edu/ccf/).

Amino acids: 500 μl frozen aliquots of sterile‐filtered L‐Arg (25 mg/ml), L‐Lys (25 mg/ml), L‐Pro (10 mg/ml), 13C615N4‐L‐Arg and 13C6 15N2‐L‐Lys can be obtained from the Duke Proteomics Core Facility.

Antibiotics: Penicillin, Streptomycin and Fungizone are recommended and can be purchased from the Duke CCF

Preparation of Media

Light Media: To a 500 ml bottle of Arg‐ and Lys‐free media, add 50 ml of dialyzed FBS, 5 ml of Pen/Strep/Fungizone and 500 μl each of Arg, Lys and Pro.

Heavy Media: To a 500 ml bottle of Arg‐ and Lys‐free media, add 50 ml of dialyzed FBS, 5 ml of Pen/Strep/Fungizone and 500μl each of 13C6 15N4 –Arg, 13C6 15N2 –Lys and Pro.

**Aliquots of heavy and light amino acids can be obtained from the Duke Proteomics Core Facility “at cost”, which is $65 for a single (500μL) aliquot of heavy/light Lys and Arg, and 2 aliquots of Pro.**

Conditioning of Cells

  1. Split cells (e.g. HEK‐293, 1:6) and plate on two 60 or 100 mm dishes containing light media. After cells have adhered (up to16h after plating), aspirate media from one dish and replace with heavy media.
     
  2. Continue to subculture light and heavy cells with the appropriate media until heavy isotope is fully (>99%) incorporated, at least 6 doublings.

Notes

  1. Fungizone is recommended in addition to Pen/Strep to prevent contamination of the media.
  2. Cells may grow slower‐than‐expected in media containing dialyzed serum.
  3. For each new heavy cell line generation, we recommend a check of isotope incorporation and a check for evidence of proline conversion in the heavy sample by mass spectrometry after the recommended number of doublings, prior to performing experimental challenge using the cells. This can be done by the DPCF using a small number of cells (~1e6) for $100.

Authored by Dr. Matthew Foster, edited by J. Will Thompson

Duke Department of Pulmonology and Duke Proteomics Core Facility.