Author:
Schober Florian A.,Atanassov Ilian,Freyer Christoph,Wredenberg Anna
Abstract
AbstractProtein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.
Reference10 articles.
1. Ong S-E, Blagoev B, Kratchmarova I et al (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
2. Ong S-E (2012) The expanding field of SILAC. Anal Bioanal Chem 404:967–976
3. Fredens J, Engholm-Keller K, Giessing A et al (2011) Quantitative proteomics by amino acid labeling in C. elegans. Nat Publ Group 8:845–847
4. Krüger M, Moser M, Ussar S et al (2008) SILAC mouse for quantitative proteomics uncovers kindlin-3 as an essential factor for red blood cell function. Cell 134:353–364
5. Sury MD, Chen J-X, Selbach M (2010) The SILAC fly allows for accurate protein quantification in vivo. Mol Cell Proteomics 9:2173–2183
Cited by
3 articles.
订阅此论文施引文献
订阅此论文施引文献，注册后可以免费订阅5篇论文的施引文献，订阅后可以查看论文全部施引文献