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Incorporation of β-actin loading control into zymography.
- Source :
-
Journal of biological methods [J Biol Methods] 2016 Nov 01; Vol. 3 (4). - Publication Year :
- 2016
-
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.<br />Competing Interests: A provisional patent application entitled “Loading Controlled Zymography-based Method for Detecting Endopeptidases” was filed by P.J. in 2014.
Details
- Language :
- English
- ISSN :
- 2326-9901
- Volume :
- 3
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Journal of biological methods
- Publication Type :
- Academic Journal
- Accession number :
- 28191477
- Full Text :
- https://doi.org/10.14440/jbm.2016.157