- Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)
- NeuCode™ SILAC
SILAC refers to labeling cultured cells with heavy amino acids for quantitative proteomic analysis. Labeling an entire proteome with heavy amino acids in vivo generates an ideal standard for quantitative proteomics. When a heavy labeled proteome is mixed with an unlabeled proteome then digested, every unlabeled peptide identified by the mass spectrometer can be quantified by its corresponding heavy peptide. In SILAC, the tryptic amino acids, arginine(R) and lysine (K), contain heavy stable isotopes, so if digesting with trypsin, every peptide is labeled. This metabolic labeling strategy has been employed by hundreds of proteomic studies (see example references below). The advantage of metabolic labeling over in vitro tagging techniques is that the heavy and unlabeled samples are mixed before sample preparation, preventing variability between preparations distorting the quantitation results. This is especially important when extensive sample preparation (e.g. isolation of an organelle) is required.
Frequently Asked Questions
Does CIL offer media, kits and reagents for SILAC?
How many samples can be tested with the SILAC Protein Quantitation Kit?
Each kit contains 500 mL media, which can isotopically label approximately 1 x 1012 cells, depending on cell type.
Do you have a protocol?
Yes, click here to download the SILAC Protein Quantitation Kit instructions for use.
Would 0.8 mM of Lysine or Arginine preloaded resins provide approximately 8 synthesis at 0.1 mM scale?
Yes, theoretically 0.8 mM of resin would provide 8 synthesis at 0.1MM scale. Final yields are always dependent on actual sequence and the efficiency of the couplings.
Singh, S.A.; Andraski, A.B.; Pieper, B. 2016. Multiple apolipoprotein kinetics measured in human HDL by high-resolution/accurate mass parallel reaction monitoring. J Lipid Res., 57(4):714-28. PMID: 26862155
Cox, T.R.; Schoof, E.M.; Gartland, A. 2015. Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism. Data Brief, 5:621-5. PMID: 26649326
McShane, A.J.; Bajrami, B.; Ramos, A.A. 2014. Targeted proteomic quantitation of the absolute expression and turnover of cystic fibrosis transmembrane conductance regulator in the apical plasma membrane. J Proteome Res., 13(11):4676-85. PMID: 25227318
Bagert, J.D.; Xie, Y.J.; Sweredoski, M.J. 2014. Quantitative, time-resolved proteomic analysis by combining bioorthogonal noncanonical amino acid tagging and pulsed stable isotope labeling by amino acids in cell culture. Mol Cell Proteomics, 13(5):1352-8. PMID: 24563536
Edfors, F.; Bostrom, T.; Forsstrom, B. 2014. Immunoproteomics using polyclonal antibodies and stable isotope-labeled affinity-purified recombinant proteins. Mol Cell Proteomics, 13(6):1611-24. PMID: 24722731
Rayavarapu, S.; Coley, W.; Cakir, E. 2013. Identification of disease specific pathways using in vivo SILAC proteomics in dystrophin deficient mdx mouse. Mol Cell Proteomics, 12(5):1061-73. PMID: 23297347
Kristensen, A.R.; Gsponer, J.; Foster, L.J. 2013. Protein synthesis rate is the predominant regulator of protein expression during differentiation. Mol Syst Biol., 9:689. PMID: 24045637
Liao, L.; McClatchy, D.B.; Park, S.K.; Xu, T.; Lu, B.; Yates III, J.R. 2008. Quantitative analysis of brain nuclear phosphoproteins identifies developmentally regulated phosphorylation events. J Proteome Res, 7(11):4743-55. PMID: 18823140
Selbach, M.; Mann, M. 2006. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nature Methods, 3, 981-983. PMID:17072306
Mann, M. 2006. Functional and quantitative proteomics using SILAC. Nat Rev Mol Cell Biol, 7, 952-958. PMID: 17139335
Amanchy, R.; Kalume. D.E.; Pandey, A. 2005. Stable isotope labeling with amino acids in cell culture (SILAC) for studying dynamics of protein abundance and posttranslational modifications. Sci STKE, 267, 1-20. PMID:15657263
Kratchmarova, I.; Blagoev. B.; Haack-Sorensen. M.; Kassem, M.; Mann, M. 2005. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science, 308, 1472-1477. PMID:15933201
Blagoev, B.; Ong, S.E.; Kratchmarova. I.; Mann. M. 2004. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nature Biotechnology, 22, 1139-1145. PMID:15314609
Ong, S.E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D.B.; Steen. H.; Pandey. A.; 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. PMID:12118079
Andrew Percy, PhD
Senior Applications Chemist - Mass Spectrometry
Dr. Andrew Percy is the Senior Applications Chemist for Mass Spectrometry. His responsibilities minimally involve overseeing product development, identifying new product market opportunities, assisting in the analysis of products for MS ‘omics applications, and providing technical support to customers.More
Proteomics Product Manager, Deuterated Reagents & Intermediates Product Manager, China Sales - Research Products
Tasha Agreste, Product Manager for the Proteomics product line, has been with CIL since 1989. During her tenure at CIL, Tasha has held various positions including customer service, sales and marketing, where she has proven to be instrumental in enhancing CIL’s customer relationships.More