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Immuno‐qPCR detection of the tandem affinity purification (TAP)‐tag as a sensitive and accurate tool suitable for large‐scale protein quantification
Author(s) -
Lind Kristina,
Norbeck Joakim
Publication year - 2007
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200700577
Subject(s) - tandem affinity purification , quantitative proteomics , protein purification , chromatography , biology , proteomics , chemistry , microbiology and biotechnology , computational biology , biochemistry , affinity chromatography , enzyme , gene
The tandem affinity purification (TAP)‐tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large‐scale protein quantification studies. We have developed an immuno‐quantitative real‐time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP‐tagged (and protein A‐tagged) proteins in yeast with a detection range between 10 7 and 10 10 molecules. The immuno‐qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)‐based large‐scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large‐scale Western blotting‐based quantification of the TAP‐tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno‐qPCR approach was demonstrated by analysis of osmo‐regulated proteins, where the 2000‐fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno‐qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP‐tag and protein A are useful in most model organisms, the immuno‐qPCR method is both generic and suitable for large‐scale studies.