Stable Isotopic Labeling by Amino Acids in Cell Culture or SILAC
SILAC1 is used to quantify protein expression differences in typically 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] for a 2 plex) 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 can be 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 are mixed after the cells are grown.
As a result, downstream processes are all subjected to the same magnitude of experimental variations, reducing the error on the final intensity ratios of the samples analyzed.
- SILAC labeling is performed in the investigators lab
- Sample mixing can be performed here after protein quantitation
Suitable Protein Complexity
|SILAC-premixed-solution||SILAC samples are mixed in the submitters lab prior to sample submission||Sample clean-up step using an extraction or precipitation technique, a dual enzymatic digestion, amino acid analysis protein quantitation on each sample when our staff mixes the samples, a long LC-MS/MS gradient run, a database search and data analysis. Grouped costs with fractions also include a strong cation fractionation or C18 reverse phase separation into 10 fractions which are run individually on a long length gradient.|
|SILAC-HL-ratios-solution||compares/quantifies heavy and light samples after amino acid analysis and subsequent mixing in our lab|
|SILAC-HL-ratios-fractions (MudPIT)||fractionation is performed after amino acid analysis and mixing in our lab; 10 fractions are analyzed by LC-MS/MS|
|SILAC-HML-ratios-solution||compares/quantifies heavy, medium and light samples after amino acid analysis and subsequent mixing in our lab|
|SILAC-HML-ratios-fractions (MudPIT)||fractionation is performed after amino acid analysis and
mixing in our lab; 10 fractions are analyzed by LC-MS/MS
|1Ong, 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.|