title: Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate.
abstract: A sensitive and selective analytical method for the determination and quantification of endogenous β-galactosidase in crude E. coli cell lysates by immunoprecipitation combined with automated microchip capillary gel electrophoresis (IP-MCGE) with laser-induced fluorescence (LIF) detection was developed. Total cell lysates were derivatized minimally with a fluorescence dye, incubated with anti-β-galactosidase antibodies, and the antigen/antibody complex was precipitated with protein G-coated magnetic beads. After capturing the complex, it was eluted from the beads under denaturing conditions and loaded directly onto a multisample microchip for analysis. The effects of antibody selection and the importance of preclearing steps were studied in detail. For quantification, an external calibration through spiking pure β-galactosidase into E. coli lysate was performed. Recovery rates of immunoprecipitation after spiking experiments and the amount of unknown endogenous β-galactosidase in E. coli lysates were determined. As proof of principle, E. coli cultures grown on nutrition media with several glucose/lactose ratios were analyzed. Differences in the expression level of β-galactosidase could be detected and measured with the developed method. Detected amounts of β-galactosidase in different culture media correlated with the β-galactosidase activities in these cultures.
authors.affiliation: Institute of Chemical Technologies and Analytics, Vienna University of Technology, Vienna, Austria.
authors.forename: Ela
authors.initials: E
authors.lastname: Herwig
authors.forename: Martina
authors.initials: M
authors.lastname: Marchetti-Deschmann
authors.forename: Christian
authors.initials: C
authors.lastname: Wenz
authors.forename: Andreas
authors.initials: A
authors.lastname: Rüfer
authors.forename: Günter
authors.initials: G
authors.lastname: Allmaier
mesh_terms: D019075:Electrophoresis, Capillary / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology*; D047468:Immunoprecipitation / Q000379:methods*; D001616:beta-Galactosidase / Q000032:analysis* / Q000378:metabolism*
chemical_list: D001616:beta-Galactosidase