SILAC Media

The Silantes SILAC Media can be obtained either as complete kit or as single components.

The Kit consists of :

   - DMEM-media. The media contains all amino acids except lysine and arginine and glutamine (c=200mM)

      (separately delivered, since this amino acid is labile)

   - FBS dialysed

   - amino acids: lysine and arginine

The kit nr below confers to the amino acid composition (heavy or light)

Notes:

1.           1.   Lys-0 and Arg-0 mean unlabeled lysine and arginine.

         Lys-4 means ²H₄-labeled lysine.

         Lys-6 and Arg-6 mean ¹³C₆-labeled lysine and ¹³C₆-labeled arginine.

         Lys-8 means ¹³C₆, ¹⁵N₂-labeled lysine.

         Arg-10 means ¹³C₆, ¹⁵N₄-labeled arginine.

    2.    DMEM: Dulbecco's Modified Eagle Medium (with glutamine(delivered separately), without arginine and lysine)

         FBS: fetal bovine serum (normal and dialyzed)

    3.    The cells should be adapted to the SILAC medium for 5 passages before they are propagated to a scale needed for

            the experiment.

    4.    Store the media at 4°C

          5.    For use mix all the components (Media, amino acids and FBS)and filtered under sterile condition.

          6.  Formulation of DMEM:  DMEM.pdf

SILAC (Stable Isotopic Labelling of Amino Acids in Cell Culture) (Ong et Mann, 2006) has been proven as a powerful technique for quantitative proteomics in cell culture. The method is robust and provides accurate results (Ong et al., 2002).

The procedure in short:

Cells are grown in culture media that are identical with exception of amino acids. The “light” culture (L) contains all the amino acids in unlabelled form and the “heavy” culture (H) contains one or two amino acids in labelled form. The labelled amino acids are usually lysine labelled with ²H (D4), ¹³C or ¹³C¹⁵N and/or arginine labelled with ¹³C or ¹³C¹⁵N. The labelled amino acids increase the molecular weight allowing identification and quantification of the proteins/peptides stemming from culture (L) and (H), respectively.

Workflow:

The following figure shows the work flow of the procedure. In order to determine quantitatively differences in the protein pattern of culture (L) and (H),

-          the cells from both cultures are mixed;

-          the proteins are isolated, then cleaved into peptides (by either LysC or trypsin), and finally

-          subjected to LS-MS analysis.

The figure shows that cells are grown in cell cultures A (light) and B (heavy), whereby culture B is labeled with e.g. ¹³C-lysine by metabolic enrichment. This is achieved by preparing a cell culture medium without lysine. The medium of cell culture A is supplemented with ¹²C₆-lysine (L) and that of B with ¹³C_6-lysine (H). Cells from both cultures are mixed in a 1:1 ratio. The proteins of interest are extracted and digested with LysC, a protease which specifically cleaves at lysines. The proteolytic cleavage creates pairs of peptides stemming from culture A and B, differing by a molecular weight of 6Da due to the molecular weight difference on end standing ¹³C₆-lysine. The ratio of the amount of light and heavy peptides is determined by mass spectrometry.

Literature:

Ong, S.E., and Mann, M. (2006). A practical recipe for stable isotope labelling by amino acids in cell culture (SILAC). Nat. Protoc. 1, 2650–2660. Ong, S.E., Blagoev, B., Kratchmarova, I., Kristensen, D.B., 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, Mol.Cell. Proteomics 1, 376–386.