We previously demonstrated microchip phosphate-affinity electrophoresis (μPAE) for activity measurement of kinases and phosphatases. In the current work, we extend the μPAE application to a protease: caspase-3 (CASP3). We designed a substrate peptide modified with both a fluorescent tag for label and a phosphate tag for separation. The CASP3 cleavage reaction split the two tags and made the fluorescent fragment migrate electrophoretically. The CASP3 activity was correlated with the amount of the migrating fragment. This assay was proven to be compatible with a crude cell lysate.
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Application of microchip phosphate-affinity electrophoresis to measurement of protease activity in complex samples.
Anal Biochem
January 2013
Bioengineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
We previously demonstrated microchip phosphate-affinity electrophoresis (μPAE) for activity measurement of kinases and phosphatases. In the current work, we extend the μPAE application to a protease: caspase-3 (CASP3). We designed a substrate peptide modified with both a fluorescent tag for label and a phosphate tag for separation.
View Article and Find Full Text PDFActivity measurement of protein kinase and protein phosphatase by microchip phosphate-affinity electrophoresis.
Anal Biochem
February 2012
Bioengineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
We previously proposed microchip-based phosphate-affinity electrophoresis (μPAE) and demonstrated its application to activity measurement of a tyrosine kinase, c-Src. In this study, we extended the μPAE application to a serine/threonine kinase, protein kinase A (PKA), and to a tyrosine phosphatase, leukocyte antigen-related protein tyrosine phosphatase (LAR PTPase). For standard peptide samples, we obtained linear calibration plots, and the limits of detection were 1.
View Article and Find Full Text PDFPhosphate-affinity electrophoresis on a microchip for determination of protein kinase activity.
Electrophoresis
October 2009
Bioengineering Laboratory, RIKEN, Saitama, Japan.
We describe microchip-based phosphate-affinity electrophoresis (microPAE) for separation of peptides aimed at determination of kinase activity. The microPAE exploits two recently published technologies: autonomous sample injection for PDMS microchips and a phosphate-specific affinity ligand, Phos-tag. We prepared a fluorescently labeled substrate peptide, specific to human c-Src, and its phosphorylated form.
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