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The ICAT reagent reacts with free cysteines in proteins in cell lysates and contains an affinity tag (biotin) and an acid cleavage site (for biotin removal). The tag is isotopically coded for a “heavy” or “light” tag, with 9 amu mass difference. After reacting 100ug control protein extract with the “light” tag and an equal amount of experimental protein extract with the “heavy” tag; the samples are mixed, trypsin digested, and cysteine-containing peptides are affinity purified on an avidin column. While ICAT has the advantage of simplifying complex extracts, it cannot detect proteins that lack cysteine. The purified, cysteine containing tryptic peptides are cleaved with trifluoroacetic acid to remove the biotin group and parent proteins are identified using LC-MS/MS. Quantitation is carried out with relevant software with the results.
Nat. Biotechonl.
1999,17:994-998
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