Product Overview :
The MMP-2 Colorimetric Drug Discovery Kit is a complete assay system designed to screen MMP-2 inhibitors using a thiopeptide as a chromogenic substrate (AcPLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5). The MMP cleavage site peptide bond is replaced by a thioester bond in the thiopeptide. Hydrolysis of this bond by an MMP produces a sulfhydryl group, which reacts with DTNB [5,5’- dithiobis(2-nitrobenzoic acid), Ellman’s reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by its absorbance at 412 nm (ε= 13,600 M-1cm-1 at pH 6.0 and above). The assays are performed in a convenient 96-well microplate format. The kit is useful to screen inhibitors of MMP-2, a potential therapeutic target. An inhibitor, NNGH12, is also included as a prototypic control inhibitor. Thiol inhibitors should not be used with this kit, as they may interfere with the colorimetric assay.
Description :
Matrix metalloproteinase-2 (MMP-2, gelatinase A, 72 kDa type IV collagenase) is a member of the MMP family of extracellular proteases. These enzymes play a role in many normal and disease states by virtue of their broad substrate specificities. Targets of MMP-2 include native and denatured collagens, fibronectin, elastin, laminin-5, pro-TNF-α, and neurocan. MMP-2 is secreted as a 72 kDa proenzyme (as measured by SDS-PAGE), and activated by cleavage to 62 and 59 kDa. MMP-2 is an important target for inhibitor screening due to its involvement in diseases such as atherosclerosis, and cancer growth, angiogenesis, and metastasis.
Storage :
Store all components except the microplate (room temperature) at -70°C for the highest stability. The MMP-2 enzyme should be handled carefully in order to retain maximal enzymatic activity. It is stable, in diluted or concentrated form, for several hours on ice. As supplied, MMP-2 enzyme is stable for at least 5 freeze/thaw cycles. To minimize the number of freeze/thaw cycles, aliquot the MMP-2 into separate tubes and store at -70°C. When setting up the assay, do not maintain diluted components at reaction temperature (e.g. 37°C) for an extended period of time prior to running the assay.
Kit Components :
96-well Clear Microplate (1/2 Volume), Room temperature
Colorimetric Assay Buffer 1 x 20ml, FORM: 50mM HEPES, 10mM CaCl2, 0.05% Brij-35, 1mM DTNB, pH7.5, STORAGE: -20°C
MMP Inhibitor 1 x 50µl, FORM: 1.3 mM in DMSO, STORAGE: -70°C
MMP Substrate 1 x 50µl, FORM: 25 mM (16.4 mg/ml) in DMSO, STORAGE: -20°C
MMP2 Enzyme (Human, Recombinant) 1 x 45.7µl, FORM: P. pastoris recombinant human MMP-2 catalytic domain (calculated MW 40 kDa), One U=100 pmol/min@ 37°C, 100 µM thiopeptide P125. STORAGE: -70°C; Avoid freeze/thaw cycles.
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