Immuno-qPCR detection of the tandem affinity purification (TAP)-tag as a sensitive and accurate tool suitable for large-scale protein quantification.
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.
Lind
Kristina
K
Department of Chemical and Biological Engineering, Molecular Biotechnology, Chalmers University of Technology, Gothenburg, Sweden.
Norbeck
Joakim
J
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012015:Reference Standards; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis*
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins