Protocol abstract
Nature Protocols 3, - 505 - 516 (2008)
Published online: 6 March 2008 | doi:10.1038/nprot.2008.2
Subject Categories: Genomics and proteomics | Spectroscopy and structural analysis
Quantitative proteomics using stable isotope labeling with amino acids in cell culture
H C Harsha1,2,3,4, Henrik Molina3,4 & Akhilesh Pandey3,4,5,6
Abstract
Stable isotope labeling with amino acids in cell culture (SILAC) is a simple in vivo labeling strategy for mass spectrometry-based quantitative proteomics. It relies on the metabolic incorporation of nonradioactive heavy isotopic forms of amino acids into cellular proteins, which can be readily distinguished in a mass spectrometer. As the samples are mixed before processing in the SILAC methodology, the sample handling errors are also minimized. Here we present protocols for using SILAC in the following types of experiments: (i) studying inducible protein complexes, (ii) identification of Tyr kinase substrates, (iii) differential membrane proteomics and (iv) studying temporal dynamics using SILAC 5-plexing. Although the overall time is largely dependent on the rate of cell growth and various sample processing steps employed, a typical SILAC experiment from start to finish, including data analysis, should take anywhere between 20 and 25 d.
- Institute of Bioinformatics, International Technology Park, Bangalore 560 066, Karnataka, India.
- Manipal University, Manipal 576104, Karnataka, India.
- McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland 21205, USA.
- Department of Biological Chemistry, Johns Hopkins University, Baltimore, Maryland 21205, USA.
- Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
- Department of Oncology, Johns Hopkins University, Baltimore, Maryland 21205, USA.
Correspondence to: Akhilesh Pandey3,4,5,6 e-mail: pandey@jhmi.edu
nature-products
A-Z product listing
- 2-Mercaptoethanol(Sigma)
- Acetonitrile(J.T. Baker)
- Agarose-conjugated antiphosphotyrosine Ab 4G10(Upstate Biotechnology)
- Colloidal coomassie staining kit(Invitrogen)
- Complete protease inhibitor cocktail tablets(Roche)
- DC protein assay kit(Bio-Rad)
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