title: Incorporation of β-actin loading control into zymography.
abstract: Gelatin zymography and immunoblot are widely used gel electrophoresis techniques to study matrix metalloproteinases-2 and -9. Each method has its advantages and disadvantages. Zymography is exquisitely sensitive but offers no loading control to ensure equal sample loading. Immunoblot is a 100-1000-fold less sensitive, but allows for the probing of a sample loading control such as β-actin to ensure accurate protein loading. In this report, we describe two simple protocols that combine gelatin zymography to study MMP-2 and -9 levels with an in-gel β-actin immunoblot loading control, thus combining sensitivity and accuracy in a single assay. The protocols incorporate the loading of molecular weight markers to demarcate MMP-2/-9 from the β-actin. The first protocol utilizes the overlay of a 10% zymography gel over a 5% Tris-Glycine separating gel from which the β-actin is transferred. The second protocol involves the direct transfer of the β-actin from a single 10% zymography gel.
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1; Cardiovascular Research Centre, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1.
authors.forename: Natasha
authors.initials: N
authors.lastname: Govindasamy
authors.affiliation: Cardiovascular Research Centre, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1; Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1.
authors.forename: MengJie
authors.initials: M
authors.lastname: Yan
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1; Cardiovascular Research Centre, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1; Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1; Mazankowski Alberta Heart Institute, University of Alberta, 3-E Katz Group-Rexall Centre for Pharmacy & Health Research, 11361-87 Avenue, Edmonton, AB, Canada, T6G E1.
authors.forename: Paul
authors.initials: P
authors.lastname: Jurasz
keywords: immunoblot; loading control; matrix metalloproteinase; zymography; β-actin