Zymography: a single-step staining method for quantitation of proteolytic activity on substrate gels

Analytical Biochemistry
T M Leber, F R Balkwill

Abstract

Zymography is an electrophoretic method for measuring proteolytic activity. The method is based on an sodium dodecyl sulfate gel impregnated with a protein substrate which is degraded by the proteases resolved during the incubation period. Coomassie blue staining of the gel reveals sites of proteolysis as white bands on a dark blue background. Within a certain range the band intensity can be related linearly to the amount of protease loaded. Although the method is widely used, crucial points concerning quantitation of proteolytic activity have not been rigorously addressed. In this report we describe a new staining protocol which converts the independent staining and destaining procedure into a single step. This leads to fast and reproducible staining of zymograms permitting reliable quantitation of proteolytic activity. As shown for proMMP-9 (type IV gelatinase-b) proteolytic activity can be quantitated in a linear manner in as little as 1 h of zymogram staining. We established the detection limit for proMMP-9 (32 pg), the linear range (below 1000 pg), and the reproducibility of the assay (coefficient of variation < 15%). This improved protocol is fast and reproducible. Its linear range of almost two log scales permits assay o...Continue Reading

Citations

Jul 1, 2009·Molecular and Cellular Biology·Peter StorzAlex Toker
Mar 16, 2011·Journal of Biomedicine & Biotechnology·Jessica TrisinaRaymond R Tjandrawinata
Apr 13, 2005·Respiration; International Review of Thoracic Diseases·Kwang Joo ParkKyi Beum Lee
Feb 18, 2003·American Journal of Respiratory and Critical Care Medicine·Christine M HartogJoerg Braun
Sep 6, 2005·American Journal of Respiratory and Critical Care Medicine·Paul T G ElkingtonJon S Friedland
Jun 19, 2007·American Journal of Respiratory Cell and Molecular Biology·Paul T ElkingtonJon S Friedland
Aug 23, 2000·The Journal of Clinical Investigation·S DiasS Rafii
Mar 3, 2005·Respiratory Research·Hester van der VaartNick H T ten Hacken
Dec 15, 2010·The Tohoku Journal of Experimental Medicine·Jin-Wook KimHo-Seong Han
Jun 8, 2013·Reproduction : the Official Journal of the Society for the Study of Fertility·L A RispoliJ L Edwards
Apr 26, 2001·Analytical Biochemistry·O TillW Linss
Mar 8, 2014·European Journal of Applied Physiology·Ryan M FrancisJeffrey T Lemmer
Jul 30, 2011·Evidence-based Complementary and Alternative Medicine : ECAM·Chiung-Chi PengRobert Y Peng
Jan 12, 2008·Clinical & Experimental Metastasis·Patrick Subarsky, Richard P Hill
Nov 12, 2005·Applied Microbiology and Biotechnology·Eva LindskogLena Häggström
Jul 21, 2009·Journal of Molecular Medicine : Official Organ of the Gesellschaft Deutscher Naturforscher Und Ärzte·Vanessa F MerinoJoão B Pesquero
Mar 18, 2008·Journal of Enzyme Inhibition and Medicinal Chemistry·Xian-Chao ChengWen-Fang Xu
Jul 10, 2014·Journal of Enzyme Inhibition and Medicinal Chemistry·Viviana De LucaClemente Capasso
Feb 24, 2016·Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada·Darren G HaskettJonathan P Vande Geest
Apr 20, 2010·Toxicon : Official Journal of the International Society on Toxinology·Loubna HamzaStefano Rufini
Dec 8, 2015·Clinical Chemistry and Laboratory Medicine : CCLM·Serena RicciAngelina Di Carlo

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