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<span style = 'font-size: large'> '''Herwig, E, Marchetti-Deschmann, M, Wenz, C, Rüfer, A and Allmaier, G''' (2011) Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate.''Biotechnol J'' '''6''':420-7</span> | |||
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|{{TableHeadingColor}} |'''Abstract''' || <protect>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.</protect> | |||
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* [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=21416610 PubMed] | |||
Online version:[http://dx.doi.org/10.1002/biot.201000453 10.1002/biot.201000453] | |||
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Revision as of 22:18, 9 August 2011
Herwig, E, Marchetti-Deschmann, M, Wenz, C, Rüfer, A and Allmaier, G (2011) Immunoprecipitation combined with microchip capillary gel electrophoresis: Detection and quantification of β-galactosidase from crude E. coli cell lysate.Biotechnol J 6:420-7
| Abstract | <protect>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. |
|- |bgcolor='#CCCCFF' nowrap |Links ||
Online version:10.1002/biot.201000453 |} </protect>
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