title: His-tag protein monitoring by a fast mix-and-measure immunoassay.
abstract: Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kreisig
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig.
authors.forename: Agneta A
authors.initials: AA
authors.lastname: Prasse
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig.
authors.forename: Kristin
authors.initials: K
authors.lastname: Zscharnack
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig.
authors.forename: Daniela
authors.initials: D
authors.lastname: Volke
authors.affiliation: Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Center for Biotechnology and Biomedicine, Leipzig University, Deutscher Platz 5, 04103 Leipzig.
authors.forename: Thole
authors.initials: T
authors.lastname: Zuchner
mesh_terms: D004926:Escherichia coli / Q000276:immunology; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006639:Histidine / Q000032:analysis* / Q000276:immunology*; D007118:Immunoassay / Q000379:methods*; D011994:Recombinant Proteins / Q000032:analysis* / Q000276:immunology*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D011994:Recombinant Proteins; D006639:Histidine