title: Application of microchip phosphate-affinity electrophoresis to measurement of protease activity in complex samples.
abstract: 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.
authors.affiliation: Bioengineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan.
authors.forename: Aishan
authors.initials: A
authors.lastname: Han
authors.forename: Kazuo
authors.initials: K
authors.lastname: Hosokawa
authors.forename: Mizuo
authors.initials: M
authors.lastname: Maeda
mesh_terms: D045744:Cell Line, Tumor; D046209:Electrophoresis, Microchip; D057075:Enzyme Assays / Q000379:methods*; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry*
chemical_list: D010710:Phosphates; D010447:Peptide Hydrolases