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.

SUBMITTER: Kreisig T 

PROVIDER: S-EPMC4085604 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature