Zymography is a well-standardized protocol for the qualitative assessment and analysis of proteinases under specified conditions. However, analysis of a large number of samples simultaneously becomes a challenge when the zymography is carried out by the usual protocol of electrophoresis. This can be overcome by assaying the matrix-degrading proteinases in substrate-impregnated gels in multiwells. Enzymes are copolymerized with 300 mL of 10% acrylamide impregnated with gelatin substrate and incubated for 16 h. The gels are then stained with Coomassie blue, destained with water, and visualized with the naked eye. The intensity; if needed can be measured with a densitometer or gel documentation system. This method has been tested for bacterial collagenases as well as some matrix-degrading metalloproteinases that were purified from rat mammary gland. It can also be used to characterize the enzymes with respect to the type and concentration of the cations required for activity and the role of other regulatory molecules that may affect the enzyme activity. The added advantage of this method is that the electrophoresis set up and electricity is not needed for the procedure.
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http://dx.doi.org/10.1007/978-1-4939-7111-4_21 | DOI Listing |
Methods Mol Biol
March 2018
Regional Medical Research Centre, Indian Council of Medical Research, Port Blair, India.
Life Sci
February 2013
College of Pharmacy, Dongguk University, Seoul 100-715, Republic of Korea.
Aims: Alpinia katsumadai was recently found in our previous study to have anti-migratory and anti-invasion activities against HT-1080 cells. However, the study did not demonstrate the exact component of Alpinia katsumadai with anti-migratory and anti-invasive activities. We tested the effects and relevant mechanism of cardamonin (CDN) on the migration and invasion of cancer cells.
View Article and Find Full Text PDFBiol Reprod
August 2010
Department of Obstetrics and Gynecology, Catholic University of Sacred Heart, Rome, Italy.
Antiphospholipid antibodies (aPL) represent an important risk factor for thrombosis and recurrent miscarriage in patients with antiphospholipid syndrome (APS). The mechanisms of aPL-mediated pregnancy failure have been researched. Previous studies demonstrated that aPL bind trophoblast cells, reducing proliferation, human chorionic gonadotrophin release, and in vitro invasiveness.
View Article and Find Full Text PDFToxicol Mech Methods
October 2012
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India.
ABSTRACT Alcoholic fibrosis and its end-stage cirrhosis occur when the rate of matrix synthesis exceeds matrix degradation. Hepatic fibroproliferation is associated with alterations of hepatic tissue inhibitors of matrix metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs/matrixins) expressions. The alteration of hepatic matrixins and TIMPs expression to disease stage and inflammatory activity underlines their potential diagnostic markers in chronic liver disease.
View Article and Find Full Text PDFJ Biochem Mol Biol Biophys
October 2002
Department of Biochemistry University of Kerala, Kariavotton, Trivandrum, Kerala 695 581, India.
Production of macrophage specific matrix metalloproteinases (MMPs) by monocyte/macrophage (mo/mphi) maintained in vitro on matrix protein substrata has been examined to study the mechanism of matrix protein dependent upregulation of macrophage specific activity. Using specific blocking reagents we have found that interaction of peripheral blood mononuclear cells (PBMC) with extracellular matrix components is crucial for its differentiation to macrophages. Multiwell zymography has shown that production of MMPs was significantly inhibited in cells maintained on fibronectin (FN) pretreated with antibodies to alpha(5), beta(1) integrins and synthetic peptide RGDS.
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