SILAC
Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful approach for high-throughput quantitative proteomics. SILAC allows highly accurate protein quantitation through metabolic encoding of whole cell proteomes using stable isotope-labeled amino acids. Since its introduction in 2002, SILAC has become increasingly popular. Because it has great advantages in quantitative applications over other approaches, SILAC has a wide range of uses such as in expression proteomics, dynamic changes of protein post-translational modifications (PTMs), interaction proteomics, protein turnover and etc.
Alfa Chemistry has global experimental and analysis abilities based on our comprehensive technical platforms and professional R&D team. We can provide you with rigorous SILAC experimental design to ensure reliable quantitative analysis results.
Principle of SILAC
The principle of SILAC is based on incorporating stable isotope-labeled amino acids, such as 13C or 15N labeled arginine or lysine, into the entire proteome during protein metabolism, specifically during the process of cell culturing. In SILAC, two populations of cells are grown in two different culture media, with the "light" medium containing amino acid(s) with the natural isotope, and the "heavy" medium containing stable isotope labeled amino acid(s). After a sufficient number of cell divisions, at least five cycles in mammalian cells, all the proteins in the cell will be isotopically labeled. Then the cell populations are experimentally manipulated and then equal amounts of labeled and unlabeled cells or protein extracts are mixed. The samples are then digested into peptides. Finally, the digested peptides are analyzed with LC-MS/MS [1].
Technical Advantages
- uLess sample is required with only a few tens of micrograms of protein per sample.
- uSILAC is an in vivo labeling method that is closer to the true state of the sample.
- uSILAC can reduce the experimental difference caused by different sample preparation and the labeling efficiency is not affected by the cracking fluid.
- uSILAC is not limited by protein properties. It is not only suitable for the analysis of whole cell proteins, but also suitable for the identification and quantification of membrane proteins, and can even be applied to the whole animal level.
- uHigh throughput, in which hundreds to thousands of proteins can be simultaneously identified and quantified.
- uThe quantitative analysis results are accurate with small variation and good repeatability.
Alfa Chemistry's Workflow
The SILAC experiment in Alfa Chemistry consists of two phases: an adaptation phase (A) and an experimental phase (B). The diagram of workflow is as follows.
During the adaptation phase, cells are grown in light and heavy SILAC media until full incorporation of the heavy amino acids in the growing cells, which confirmed by MS analysis.
During the experimental phase, the cell populations are subjected to different treatments according to the aim of the study, and then combined equally prior to subsequent optional subcellular organelle purification, cell lysis, protein extraction, and protein digestion. Finally, the samples are analyzed with LC-MS/MS. The identification and quantification of peptides is accomplished with quantitation software such as MaxQuant.
※ Abbreviations: AAs, Amino acids. K0/R0, 12C/14N light amino acids. K4/R6, D4-lysine/13C6-arginine. K8/R10, 13C6,15N2-lysine+ 13C6,15N4-arginine
Alfa Chemistry's SILAC Service
- Cell culture and cell labeling
- Labeling efficiency measurement
- Protein identification and quantification
- Data analysis
- Cell lysis, protein extraction, trypsin digestion, peptide fractionation
- LC-MS/MS analysis
- Bioinformatics analysis
- Detailed report
Ordering Process
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- Superior customer service
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Reference
- Chen X., et al. Quantitative proteomics using SILAC: Principles, applications, and developments[J]. Proteomics, 2015, 15(18): 3175-3192.
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