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Development of Deuterated-Leucine Labeling with Immunoprecipitation to Analyze Cellular Protein Complex

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Author(s): Shufang Liang | Shufang Liang | Xuejiao Xu | Haojie Lu | Pengyuan Yang

Journal: Journal of Proteomics & Bioinformatics
ISSN 0974-276X

Volume: 01;
Issue: 06;
Start page: 293;
Date: 2008;
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Keywords: deuterated-leucine (Leu-d3) labeling | stable isotope labeling by amino acids in cell culture (SILAC) | mass spectrometry | immunoprecipitation | protein complex

ABSTRACT
The deuterated-leucine (Leu-d3) labeling is one kind of stable isotope labeling by amino acids in cell culture (SILAC), which has been widely used to compare and quantify protein relative expression. We expanded an integrated immunoprecipitation (IP) coupled with SILAC approach (SILAC-IP) to differentiate the specific binding partners associated with a bait protein in two populations of cells. By this SILAC-IP strategy, the identified specific-binding proteins were quantified by tracking pairs of Leu-d3 labeled and unlabeled peptides from the mass spectra, which could differentiate specific-binding proteins from nonspecific partners in high confidence. We applied SILAC-IP method to differentiate specific-binding proteins associated with 14-3-3â in human hepatocellular carcinoma cell line QGY7703 between those in the liver cell line QSG7701. The proteins including HSP86, SKB1hs, GADPH and MEP50 were identified to associate with 14-3-3â in QGY7703 with high binding level than in QSG7701 cells.
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Tango Jona
Tangokurs Rapperswil-Jona