title: Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers.
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.
authors.affiliation: Institute of Biochemistry and Biology, Biotechnology, University of Potsdam, Golm, Germany.
authors.forename: Katjana
authors.initials: K
authors.lastname: Daskalow
authors.forename: Prisca
authors.initials: P
authors.lastname: Boisguerin
authors.forename: Burkhard
authors.initials: B
authors.lastname: Jandrig
authors.forename: Rudolf
authors.initials: R
authors.lastname: Volkmer
authors.forename: Burkhard
authors.initials: B
authors.lastname: Micheel
authors.forename: Jörg A
authors.initials: JA
authors.lastname: Schenk
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology*; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000906:Antibodies; D011993:Recombinant Fusion Proteins