Real-time zymography and reverse zymography: a method for detecting activities of matrix metalloproteinases and their inhibitors using FITC-labeled collagen and casein as substrates.
Zymography and reverse zymography are widely used techniques for identifying the proteolytic activity of enzymes and the presence of protease inhibitors in polyacrylamide gels. In the current studies, we utilized a fluorescein-isothiocyanate-labeled substrate to develop novel zymographic and reverse zymographic methods for detecting matrix metalloproteinases and tissue inhibitors of the metalloproteinases, respectively. Using a transilluminator, the results can be observed visually without stopping the enzymatic reaction. For this reason, we have named these methods real-time zymography and real-time reverse zymography. These methods have the following advantages compared with conventional protocols: (1) because the reaction can be repeatedly monitored on the polyacrylamide gels, optimization of the incubation time can be achieved without preliminary analyses; (2) higher sensitivity is achieved with a lower amount of substrate than with conventional methods; (3) a semi-quantitative analysis of matrix metalloproteinases is possible. An additional advantage of the real-time reverse zymography is that, because the fluorescence detection is specific for substrate digestion, the inhibitor bands can be easily distinguished from contaminating proteins.
Hattori
Shunji
S
Nippi Research Institute of Biomatrix, Tokyo, Adachi-ku, 120-8601, Japan. shuhatt@bekkoame.ne.jp
Fujisaki
Hitomi
H
Kiriyama
Tomomi
T
Yokoyama
Tsukao
T
Irie
Shinkichi
S
D002364:Caseins / Q000737:chemistry / Q000378:metabolism*; D004796:Clinical Enzyme Tests / Q000379:methods*; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry*; D020782:Matrix Metalloproteinases / Q000032:analysis*; D011489:Protein Denaturation; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D013194:Staining and Labeling; D013379:Substrate Specificity; D019714:Tissue Inhibitor of Metalloproteinases / Q000032:analysis*
D002364:Caseins; D019714:Tissue Inhibitor of Metalloproteinases; D012710:Serum Albumin, Bovine; D003094:Collagen; D020782:Matrix Metalloproteinases; D016650:Fluorescein-5-isothiocyanate