A Practical Formulation Guide for SILAC Technology
What Is SILAC Technology?
Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology involves the metabolic incorporation of stable isotope-labeled amino acids into cellular proteins during cell culture. SILAC is a simple, stable and powerful method in quantitative proteomics based on mass spectrometry (MS). In quantitative proteomics, SILAC can provide accurate relative quantification without any chemical derivatization or other operations, and is widely used in research fields such as cell biology, biochemistry, and pharmacology.
SILAC Medium Formulation
The formulation of SILAC media is a critical step in implementing this technology effectively. The SILAC medium should contain all essential amino acids, with one or more of them replaced with the corresponding heavy isotope-labeled amino acid.
- Light medium
Normal culture medium such as DMEM (Dulbecco's Modified Eagle Medium), RPMI 1640 (Roswell Park Memorial Institute), and MEM (Minimum Essential Medium). - Heavy Medium
Use 13C or 15N-labeled amino acids, such as leucine (13C-Leu), lysine (13C-Lys) and methionine (13C-Met). - Other ingredients
Include other nutrients required for cell growth, such as dialyzed fetal bovine serum and specific supplements.
Fig 1. SILAC protocol. [1]
- Cell culture
The cells are divided into two parts, one part is cultured in light-labeled medium and the other part is cultured in heavy-labeled medium. After 6-7 doubling cycles, the amino acids labeled with stable isotopes in cells cultured in heavy-labeled medium can be completely incorporated into newly synthesized proteins. - Notes
Labeling efficiency detection: It is recommended to perform labeling efficiency detection after the cells have undergone multiple doubling cycles to ensure that the labeling efficiency meets the SILAC technical requirements.
Experimental operation: In order to more conveniently perform drug treatment or conditional stimulation, it is recommended to perform experiments in light-labeled cultured cells.
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Choice of Isotope-Labeled Amino Acids in SILAC
The choice of isotope-labeled amino acids in SILAC technology is crucial for accurate and specific protein labeling. Popular choices include heavy isotope-labeled forms of lysine (13C6, 15N2-lysine) and arginine (13C6, 15N4-arginine), which can be incorporated into newly synthesized proteins during cell culture. The following table lists the amino acid composition and quality differences used in SILAC culture media.
More Isotope-Labeled Amino Acids from Alfa Chemistry
References
- Shao-En Ong, et al. Nature protocols, 20116, 1(6), 2650-2660.
- Guoan Zhang, et al. Phospho-Proteomics: Methods and Protocols, 2009, 527, 79-92.
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