SILAC Reagents for Quantitative Proteomics in Cell Culture

SILAC, which stands for Stable Isotope Labeling of Amino Acids in Cell Culture, is an effective method for identifying and quantifying relative differences in complex protein samples. The procedure includes the metabolic incorporation of “heavy” ¹³C- or ¹⁵N-labeled amino acids into proteins in vivo, which is then followed by mass spectrometry (MS) for comprehensive and expedited protein identification, characterization, and quantification.SILAC enables the comparison of different cellular states or time points under identical conditions, making it a powerful tool for studying cell signaling, post-translational modifications, protein-protein interactions, and gene expression regulation.

In-vitro SILAC Proteomics Media and Kits

The SILAC method has been proven to be a powerful technique for quantitative proteomics in cell culture. The SILAC medium is employed in conjunction with isotopically labeled amino acids to achieve differential isotopic labeling of cells for SILAC, followed by analysis with mass spectrometry (MS). The method is robust and provides accurate results.[1]

SILAC Reagents for in-vitro cell studies

Silantes supplies a comprehensive portfolio of essential materials for the SILAC experiment, including individual products or kits consisting of the necessary components for a specific amino acid combination, including:

SILAC Amino Acids

SILAC amino acids are essential reagents of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) application and analysis. The heavy and light amino acids, such as L-lysine and L-arginine (labeled and unlabeled), are used for analysis of protein expression by mass spectrometry.

Selected products in this field (complete product range at bottom of page):

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SILAC Media

Media for SILAC is optimized to analyze protein expression by mass spectrometry (MS). Silantes provides SILAC complete media, such as:

SILAC DMEM: Dulbecco’s Modified Eagle Medium (DMEM) is a specialized media that is deficient in certain amino acids, such as L-lysine and L-arginine.

SILAC RPMI: Roswell Park Memorial Institute Medium (RPMI) is a specialized media that is deficient in certain amino acids, such as L-lysine and L-arginine.

SILAC FBS: A dialyzed fetal bovine serum specifically designed for use in SILAC experiments.

SILAC Kits

SILAC kits are specialized ready-to-use aliquots that contain essential components, such as amino acids and media for SILAC experiments. These kits typically contain SILAC amino acids, SILAC media (such as SILAC DMEM or SILAC RPMI), and SILAC dialyzed FBS.

SILAM for in-vivo studies

The SILAM (Stable Isotope Labeling of Amino Acids of Mammals) method is a well-established in-vivo labeling approach for relative tissue protein and metabolite quantification by mass spectrometry.

Both methods differ primarily in that SILAC is used for in-vitro cell culture experiments, while SILAM is used for in-vivo protein labeling and quantification in living organisms, particularly mammals.

This approach has been successfully applied to the analysis of mouse and fly organs. Both methods allow precise quantitative proteomic analysis by stable isotope labeling of amino acids.

Selected products in this field (complete product range at bottom of page):

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In-vitro SILAC Protein Quantitation Workflow

The principle of SILAC is based on the metabolic incorporation of stable isotope labeled amino acids into the entire cell proteome. In this technique, two populations of cells are grown in separate culture media. The first population is grown in a “light” medium containing amino acids with natural isotopes, while the second population is grown in a “heavy” medium containing stable isotope-labeled amino acids. After a sufficient number of cell divisions, all proteins from the cells grown in the “heavy” medium contain amino acids in the isotopically labeled (heavy) state.

When analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), quantification in SILAC is based on determining the ratio of introduced isotopically labeled peptides to their unlabeled counterparts. The signal intensities of the light and heavy samples allow a quantitative comparison of their relative abundances in the mixture. This approach allows accurate measurement of changes in protein expression levels between the two cell populations by directly comparing the intensities of the corresponding light and heavy peptide pairs.

The steps below and figure 1 illustrate the workflow of the SILAC metabolic labeling for quantitative determination of the differences in the protein patterns of two different cultures:

Step 1: Culture A (“light”) is supplemented with unlabeled amino acids, while culture B (“heavy) is supplemented with labeled 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 of peptides stemming from culture A and B, differing by a molecular weight of 6 Da due to the molecular weight difference of the terminal 13C6-lysine. The ratio of the amount of “light” and “heavy” peptides is determined by mass spectrometry.

Products for In-vitro SILAC Quantitative Proteomics

Silantes provides all isotope labeling reagents and kits required for a SILAC experiment. Each component is in a prepared sterile solution and ready to use for stable isotope labeling with amino acids. The components are available as single products or in a kit.

SILAC DMEM

Dulbecco’s Modified Eagle Medium (DMEM) formulation, optimized for SILAC with the necessary specifications for mass spectrometry analysis, w/o L-Gln, L-Arg, L-Lys, solution.

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SILAC RPMI

Roswell Park Memorial Institute Medium (RPMI) formulation for SILAC, w/o L-Gln, L- Arg, L-Lys, solution.

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SILAC FBS

SILAC FBS is dialyzed fetal bovine serum used in SILAC experiments for quantitative proteomics analysis in research. SILAC FBS products are available in different volumes.

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SILAC Kits

A complete set of products for SILAC-based protein quantitation, including kits, reagents, and media, aimed at providing accurate protein quantitation with mass spectrometry.

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[1] 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.