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In-vitro SILAC
In vitro SILAC (Stable Isotope Labelling of Amino Acids in Cell Culture) has been proven a powerful
technique for quantitative proteomics in cell culture. The method is robust and provides accurate results. 1
The in-vitro SILAC workflow:
Figure 1 shows the workflow of the SILAC procedure toquantitatively determine differences in the protein pattern of
two cultures:
Step 1: Culture A (“light”) is supplemented with unlabelledamino acids, whereby culture B (“heavy) is supplemented withlabelled amino acids. As an example, in culture B, the 12C6-lysine
is substituted by 13C6-lysine.
Step 2: Cells from both cultures are mixed in a 1:1 ratio. The
proteins are isolated and digested with Lys-C, a protease which
specifically cleaves at lysines.
Step 3: The proteolytic cleavage creates corresponding pairs ofpeptides stemming from culture A and B, differing by amolecular weight of 6 Da due to the molecular weight
difference of the terminal 13C6-lysine. The ratio of the amountof “light” and “heavy” peptides is determined by mass
spectrometry.
Figure 1: In-vitro SILAC workflow

Silantes Components for in-vitro SILAC Experiments
Silantes offers all components that are necessary for a SILAC
experiment. Each component is in a prepared sterile solution andready for use. The components are available as individual products
or in a kit. Each kit consists of:▪

2 х 500 mL Silantes SILAC DMEM or RPMI media free of theamino acids lysine and arginine▪

2 х 50 mL Silantes dialyzed FBS▪

Unlabelled L-lysine and L-arginine▪

SILAC amino acids L-lysine and L-arginine

1Ong, 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.

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Scan the QR-Code to see our productcatalogue of in-vitro SILAC components.

In-vitro SILAC
High Quality of Silantes SILAC Components
The SILAC amino acids are available in all isotopic combinations. We guarantee an isotopic enrichment of> 98 atom % with a chemical purity of > 95 %. The isotopic purity is tested by mass spectrometry, whereas
the chemical purity is tested by HPLC.
MDC K II wt

Figure 2 shows the growth

6,0E +06

kinetics

model

5,4E +06

mammalian cell line using

4,8E +06

ofa

5,7E +06
5,1E +06
4,5E +06

Silantes SILAC media and4,2E +06

different labelling patterns

3,6E +06
C ells / mlof the SILAC L-lysines.

3,9E +06+ L ys+ L ys C 6N2+ L ys C 6+ L ys N2- L ys3,3E +06
3,0E +06
2,7E +06

The experiment shows that

2,4E +06

the cells grow well on the

1,8E +06

Silantes SILAC components.

1,2E +06

2,1E +06
1,5E +06
9,0E +05
6,0E +05
3,0E +05
0,0E +00-12012

24

36

48

60

72

84

96

108

120132T ime [h]Figure 2: Kinetics of MDCK cells on Silantes SILAC media

Figure3incorporationshowsoftheC6-13

lysine in an actin peptide(molecular weight = 586Da) during the preparationof the “heavy” culture for aSILAC experiment
A comparison of the 586
Da peak at t = 0 hours
stemming

Figure 3: Incorporation of Silantes Lys-6

fromtheunlabelled actin peptide
and the 589 Da peak at t = 8 days stemming from the corresponding labelled actin peptide indicates thatthe cell culture is fully labeled after 8 days (4 passages). That the nominal difference of the peaks is 3 Da
(and not 6 Da) is due to the fact that the ratio m/z (x-coordinate) is 2 (instead of 1).

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