Silac

Information about Silac

The principle of SILAC. Cells differentially labeled by growing them in light medium with normal arginine (Arg-0, grey colour) or medium with heavy arginine (Arg-6, red colour). Metabolic incorporation of the amino acids into the proteins results in a mass shift of the corresponding peptides. This mass shift can be detected by a mass spectrometer as indicated by the depicted mass spectra. When both samples are combined, the ratio of peak intensities in the mass spectrum reflects the relative protein abundance. In this example, the labeled protein has the same abundance in both samples (ratio 1).

SILAC (Stable isotope labelling with amino acids in cell culture) is a mass spectrometry-based technique developed in the group of Matthias Mann to detect differences in protein abundance between two samples (Ong et al., 2002). It is one of the most popular methods for quantitative proteomics. Two populations of cells are cultivated in cell culture. One of the cell populations is fed with growth medium containing normal amino acids. In contrast, the growth medium of the second cell population contains amino acids labeled with stable (non-radioactive) heavy isotopes. For example, the medium can contain arginine labeled with six carbon-13 atoms (¹³C) instead of the normal carbon-12 (¹²C). When the cells are growing in this medium, they incorporate the heavy arginine into all of their proteins. Therefore, all of the arginine containing peptides are now 6 Da heavier than their normal counterparts. The trick is that the proteins from both cell populations can be combined and analyzed together by mass spectrometry. Pairs of chemically identical peptides of different stable-isotope composition can be differentiated in a mass spectrometer owing to their mass difference. The ratio of peak intensities in the mass spectrum for such peptide pairs accurately reflects the abundance ratio for the two proteins. SILAC has emerged as a very powerful method to study cell signaling and protein-protein interaction.

References

Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, 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". Molecular & Cell Proteomics 1: 376-86.
Ong SE, Kratchmarova I, Mann M (2003). "Properties of 13C-substituted arginine in stable isotope labeling by amino acids in cell culture (SILAC)". Journal of Proteome Research 2: 173-81.
Ong SE, Mann M (2006). "A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC)". Nature Protocols 1: 2650-60.
Ong SE, Mann M (2007). "Stable isotope labeling by amino acids in cell culture for quantitative proteomics". Methods in Molecular Biology 359: 37-52.

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Mass spectrometry (previously called mass spectroscopy ()^[1] or informally, "mass-spec" and MS) is an analytical technique used to measure the mass-to-charge ratio of ions.
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Matthias Mann (born 1959) is a scientist in the area of mass spectrometry and proteomics. Born 1959 in Germany he studied mathematics and physics at the University of Goettingen. He received his Ph.D.
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The aim of quantitative proteomics is to obtain quantitative information about all proteins in a sample. Rather than just providing lists of proteins identified in a certain sample, quantitative proteomics yields information about differences between samples.
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Cell culture is the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells.
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growth medium or culture medium is a substance in which microorganisms or cells can grow.^[1] There are different types of media for growing different types of cells.
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amino acid is a molecule that contains both amine and carboxyl functional groups. In biochemistry, this term refers to alpha-amino acids with the general formula H₂NCHRCOOH, where R is an organic substituent.
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Isotopes are any of the several different forms of an element each having different atomic mass (mass number). Isotopes of an element have nuclei with the same number of protons (the same atomic number) but different numbers of neutrons.
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Arginine (abbreviated as Arg or R)^[1] is an α-amino acid. The L -form is one of the 20 most common natural amino acids. Its codons are CGU, CGC, CGA and CGG.
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Carbon-13 (¹³C)is a natural, stable isotope of carbon and one of the environmental isotopes. It makes up about 1.1% of all natural carbon on Earth.

Detection by NMR spectroscopy

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Carbon-12 is the more abundant of the two stable isotopes of the element carbon, accounting for 98.89% of carbon; it contains 6 protons, 6 neutrons and 6 electrons.
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Cell signaling is part of a complex system of communication that governs basic cellular activities and coordinates cell actions. The ability of cells to perceive and correctly respond to their microenvironment is the basis of development, tissue repair, and immunity as well as
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Protein-protein interactions refer to the association of protein molecules and the study of these associations from the perspective of biochemistry, signal transduction and networks.

The interactions between proteins are important for many biological functions.
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Proteomics is the large-scale study of proteins, particularly their structures and functions.^[1]^[2] Proteins are vital parts of living organisms, as they are the main components of the physiological pathways of cells.
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