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  • Title: Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate.
    Author: Herwig E, Marchetti-Deschmann M, Wenz C, Rüfer A, Allmaier G.
    Journal: Biotechnol J; 2011 Apr; 6(4):420-7. PubMed ID: 21416610.
    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.
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