Stable Isotopic Labeling by Amino Acids in Cell Culture or SILAC
SILAC (1) is used to quantify protein expression differences in up to 2 or 3 samples from cells. Cells are grown in identical conditions, with one cell line incorporating heavy isotopic amino acids (usually Arginine [U-13C6, 15N4] and Lys [U-13C6]) thereby taking advantage of the normal metabolic machinery of the cell to label the proteins. The light and heavy amino acids are chemically identical and thus co-elute in SDS PAGE and HPLC separation. But, since the peptides are isotopically distinct, the light and heavy labeled peptides are clearly distinguishable by mass spectrometry. A small percentage of the sample is removed early on to check incorporation of the heavy amino acids and a growth of 8-10 doubling times is recommended for a high incorporation. Samples should be mixed immediately after the cells are grown.
- Recommended sample amount is 50 to 100μg if the CEX fractionation is performed
- 0.5 to 5μg is recommended for a single LC-MS/MS run
*SILAC Labeling is performed in the investigators lab
- Ong, S., Blagoev, B., Kratchmarova, I., Kristensen, D., Steen, H., Pandey, A., and Mann, M. (2002) Stable Isotope Labeling by Amino Acids in Cell Culture, SILAC, as a Simple and Accurate Approach to Expression Proteomics. Molecular & Cellular Proteomics, 1(5):376-386.