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Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers

Item Type:Article
Title:Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers
Creators Name:Daskalow, K. and Boisguerin, P. and Jandrig, B. and Volkmer, R. and Micheel, B. and Schenk, J.A.
Abstract:Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15-meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.
Keywords:Monoclonal antibody, Hybridoma, Molecular weight standard, Substitution analysis, Animals, Mice
Source:Bioscience, Biotechnology, and Biochemistry
Publisher:Japan Society for Bioscience, Biotechnology, and Agrochemistry
Page Range:346-351
Date:February 2008
Official Publication:https://doi.org/10.1271/bbb.70406
PubMed:View item in PubMed

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