Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of ß-galactosidase from crude E. coli cell lysate.

ABSTRACT

A sensitive and selective analytical method for the determination and quantification of endogenous ß-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-ß-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 ß-galactosidase into E. coli lysate was performed. Recovery rates of immunoprecipitation after spiking experiments and the amount of unknown endogenous ß-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 ß-galactosidase could be detected and measured with the developed method. Detected amounts of ß-galactosidase in different culture media correlated with the ß-galactosidase activities in these cultures.