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MMP-2 proenzyme (human fibroblasts)

 
ALX-200-419-C005 5 µg 239.00 USD
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Product Specification

Alternative Name:Matrix metalloproteinase 2, Gelatinase A, 72 kDa Type IV collagenase
 
MW:~72kDa
 
Source:Isolated from human rheumatoid synovial fibroblasts. Requires activation.
 
EC:3.4.24.24
 
UniProt ID:P08253
 
Formulation:Liquid. In 50mM TRIS-HCl, pH 7.0, containing 200mM NaCl, 5mM CaCl2, 1µM ZnCl2, 0.05% BRIJ 35 and 0.05% sodium azide.
 
Purity:≥90% (SDS-PAGE, Western blot)
 
Purity Detail:No other MMP contaminants are detectable.
 
Specific Activity:≥850mU/mg protein (Y. Masui, et al.; Biochem. Med. 17, 215 (1977)). One unit is defined as the amount of enzyme that hydrolyzes 1µmol Dnp-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg-OH per min. at 37°C, pH 7.0.
 
Application Notes:Immunogen for antibody generation, control in immunoassays and for characterizing interactions with MMP inhibitors.
 
Shipping:Shipped on Dry Ice
 
Long Term Storage:-80°C
 
Handling:Avoid freeze/thaw cycles.
 
Technical Info/Product Notes:Activity: Specific activity can be assayed with the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) (Masui et al.). Substrate concentration should be 0.5mg/ml in 50mM TRIS-HCl, pH 7.0, 200mM NaCl, 5mM CaCl2, 1µM ZnCl2, 0.05% sodium azide, 0.05% BRIJ35, containing 0.05mg/ml albumine. One unit MMP catalyzes the hydrolysis of 1µmol Dnp-peptide/min. at 37°C and pH 7.0. Alternatively the fluorogenic substrate (7-Methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-N-β-Dnp-L-(α,β-diaminopropionyl)Ala-Arg-NH2 (Knight et al. 1992) can be used. Hydrolysis of the Gly-Leu bond separates the highly fluorescent (7-Methoxycoumarin-4-yl)acetyl group from the 2,4-dinitrophenyl resulting in an increase of fluorogenic intensity. The Km value for the gelatinase A is 7.0x105M-1s-1. Substrate should be kept as a 9.15mM stock solution in DMSO (10mg/ml). In the assay the substrate concentration should be ~25µM. The assay can be performed in a 96-well microtiter plate (100/200µl per well) suitable for fluorogenic measurements (Ex 328 nm; Em 393 nm).

Activation: Requires activation by 2mM (final concentration) APMA or 1mM mersalic acid for 60-120 min. at 37°C. We do not recommend to use trypsin for activation! Do not dilute enzyme for activation!

Inhibitors: Activated enzyme is inhibited by tissue inhibitors of matrix metalloproteinase-2 (TIMP-2) and by chelators of divalent cations like EDTA or o-phenanthroline.
 

Product Literature References

Activation of progelatinase A and progelatinase A/TIMP-2 complex by membrane type 2-matrix metalloproteinase: H. Kolkenbrock, et al.; Biol. Chem. 378, 71 (1997), Abstract;
A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett. 296, 263 (1992), Abstract;
Synthetic substrates for vertebrate collagenase: Y. Masui, et al.; Biochem. Med. 17, 215 (1977), Abstract;

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