Abstract：
:Although differences in protein staining intensity can often be visualized by difference gel electrophoresis, abundant proteins can obscure less abundant proteins, and quantification of post-translational modifications is difficult. We present a protocol for quantifying changes in the abundance of a specific protein or changes in specific modifications of a protein using in-gel stable isotope labeling. In this protocol protein extracts from any source treated under two experimental conditions are resolved in two separate lanes by gel electrophoresis. Parallel gel regions of interest are reacted separately with either light or heavy isotope-labeled reagents, and the gel slices are then combined and digested with proteases. The resulting peptides are then analyzed by liquid chromatography/mass spectrometry (LC/MS) to determine relative abundance of light- and heavy-isotope lysine-containing peptide pairs and analyzed by LC/MS/MS for identification of sequence and modifications. This protocol should take approximately 24-26 h to complete, including the incubation time for proteolytic digestion. Additional time will be needed for data analysis and interpretation.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Asara JM,Zhang X,Zheng B,Maroney LA,Christofk HR,Wu N,Cantley LCdoi
10.1038/nprot.2006.7subject
Has Abstractpub_date
2006-01-01 00:00:00pages
46-51issue
1eissn
1754-2189issn
1750-2799pii
nprot.2006.7journal_volume
1pub_type
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