SILAC Protein Quantitation Kits

Thermo Scientific SILAC Protein Quantitation Kits are used for the specific analysis of protein expression by mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC).

Stable isotope labeling with amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. This approach entails the in vivo metabolic incorporation of "heavy" ¹³C- or ¹⁵N-labeled amino acids into proteins followed by mass spectrometry (MS) for the accelerated and comprehensive identification, characterization and quantitation of proteins.

Highlights:

• Efficient – 100% label incorporation into proteins of living cells
• Reproducible – eliminates intra-experimental variability caused by differential sample preparation
• Flexible – media deficient in both L-lysine and L-arginine, allowing for more complete proteome coverage through dual amino acid isotope labeling
• Compatible – label proteins expressed in a wide variety of mammalian cell lines adapted to grow in DMEM, RPMI-1640 or DMEM:F12 medium, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3, Jurkat and others
• High-quality supplements – heavy amino acids with >99% isotope purity; dialyzed FBS tested to ensure that it is sterile, endotoxin-free, and cell culture compatible

Applications:

• Quantitative analysis of relative changes in protein abundance from different cell treatments
• Quantitative analysis of proteins for which there are no antibodies available
• Protein expression profiling of normal vs. disease cells
• Identification and quantification of hundreds to thousands of proteins in a single experiment

Product Details:

Example SILAC workflow. Stable isotope labeling with amino acids in cell culture (SILAC) requires growing mammalian cells in specialized media deficient in lysine and arginine. This deficiency is compensated by adding light or heavy forms of the missing amino acids; i.e., 12C6 and 13C6 L-lysine, respectively. A typical experiment involves growing one cell population in medium containing light amino acids (control), while the other population is grown in the presence of heavy amino acids (experimental). The heavy and light amino acids are incorporated into proteins through natural cellular protein synthesis. After alteration of the proteome in one sample through chemical treatment or genetic manipulation, equal amounts of protein from both cell populations are then combined, separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and digested with trypsin before MS analysis.

Because peptides labeled with heavy and light amino acids are chemically identical, they co-elute during reverse-phase column pre-fractionation and therefore are detected simultaneously during MS analysis. The relative peak intensities of multiple isotopically distinct peptides from each protein are then used to determine the average change in protein abundance in the treated sample.

Each kit includes all necessary reagents to isotopically label cells with ¹³C₆ L-lysine-2HCl, including media, heavy and light amino acids and dialyzed serum. Heavy L-arginine-HCl derivatives (Part No. 88210, 89990) are available separately and can be combined with Pierce SILAC Protein Quantitation Kits to enhance peptide isotope label coverage. Kits are compatible with mammalian cell lines adapted to grow in either DMEM or RPMI 1640 media. Kits with specialized media formulations are also available for human and murine stem cell lines. When combined with Thermo Scientific Protein/Peptide Sample Enrichment Products, Pierce SILAC Protein Quantitation Kits also enable MS analysis of low-abundance proteins such as cell surface proteins, organelle-specific proteins and post-translational protein modifications such as phosphorylation or glycosylation.

Presented Posters about SILAC:

• Characterization of human mesenchymal stem cell neuronal differentiation by SILAC and high content screening analysis
• SILAC studies of murine embryonic stem cell differentiation into muscle
• Quantitation of chemical and genetic alteration of the proteome using stable isotope labeling with amino acids in cell culture (SILAC)
• Protein quantitation of p53-regulated proteins using stable isotope labeling with amino acids in cell culture (SILAC)
• Using stable isotope labeling with amino acids in cell culture (SILAC) to quantitate exportin/CRM1-dependent protein transport in nuclear extracts

SILAC Literature References:

1. Amanchy, R. et al. (2005). Science STKE 267: 1-20.
2. Blagoev, B., et al. (2004). Nat Biotechnol 22(9): 1139-45.
3. Everley, P. et al. as presented at American Society for Biochemistry and Molecular Biology. MCP Papers in Press. Published on July 11, 2007 as Manuscript M700057-MCP200.
4. Kratchmarova, I., et al. (2005). Science 308(5727): 1472-7.
5. Mann, M. (2006). Nat Rev Mol Cell Biol 7(12): 952-8.
6. Ong, S. E., et al. (2002). Mol Cell Proteomics 1(5): 376-86.
7. Selbach, M. and Mann, M. (2006). Nat. Methods 3(12): 981-3.

Related Resources:

SILAC Protein Quantitation Kit FAQ
Tech Tip #60: Prepare SILAC peptides using the In-Gel Tryptic Digestion Kit
Review of Quantitative Proteomics
Service Providers that use our SILAC Reagents

Related Products:

Heavy and Light Amino Acids for SILAC – heavy and/or light arginine, lysine, leucine and proline
SILAC Media and Dialyzed FBS – amino acid-deficient SILAC media and components
Silver Stain for Mass Spectrometry – exceptional sensitivity and recoverability for MS
In-Gel Trypsin Digest Kit – optimized kit for protein MS sample preparation