Application of microchip phosphate-affinity electrophoresis to measurement of protease activity in complex samples

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.