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MMP-14 prodomain (catalytic domain) (human), (recombinant) (His-tag)

 
ALX-201-099-C010 10 µg 479.00 USD
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Product Specification

Alternative Name:Matrix metalloproteinase 14, MT-1 MMP, Membrane-type 1 matrix metalloproteinase
 
MW:~31kDa
 
Source:Produced in E. coli. Prodomain (catalytic domain) of human MMP-14 proenzyme (aa 1-265) is fused to a His-tag (Leu-Val-Thr-(His)6).
 
EC:3.4.24.-
 
UniProt ID:P50281
 
Concentration:0.2mg/ml
 
Formulation:Liquid. In 50mM TRIS-HCl, pH 7.5, containing 150mM NaCl and 5mMCaCl2.
 
Purity:≥90% (SDS-PAGE)
 
Specific Activity:≥60mU/mg protein after trypsin activation. One unit is defined as the amount of enzyme that hydrolyzes 1µmol Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 per min. at 37°C, pH 7.5.
 
Application Notes:Active catalytic domain MMP-14 is used to study the activation of MMP-2 (progelatinase A) and the degradation of proteins of the extracellular matrix, including fibrillar collagens. MMP-14 is inhibited by tissue inhibitors of MMP-2 and MMP-3 and by chelators of divalent cations as EDTA or o-phenanthroline.
 
Shipping:Shipped on Dry Ice
 
Short Term Storage:-20°C
 
Long Term Storage:-80°C
 
Use/Stability:Stable for several weeks when stored at -20°C.
 
Handling:Avoid freeze/thaw cycles.
 
Protocol:Activation of MMP-14 proenzyme and measurement of catalytic activity
1 Preparation and stability of solutions:

Activation buffer: 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2. Store at -20°C.
Trypsin solution: 50µg TPCK-trypsin per ml activation buffer. Store in aliquots at -20°C.
Aprotinin solution: 1mg aprotinin per ml activation buffer. Store at -20°C.
Peptide hydrolysis buffer: 50mM TRIS-HCl, pH 7.5, 150mM NaCl, 5mM CaCl2, 0.025% Brij 35. Store at +4°C for several weeks.
Stock solution of peptide substrate: 100µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 in 20% DMSO. Store at -20°C.
Stock solution of unquenched peptide: 10µM solution of Mca-Pro-Leu-NH2 in 20% DMSO. Store at -20°C.

2 Activation:
An aliquot of 5µg MMP-14 proenzyme is mixed with 1µl trypsin solution in 100µl avtivation buffer and incubated for 12 min. at 25°C. Thereafter trypsin is inhibited by addition of 1µl aprotinin solution.

3 Assay protocol:

The activity of MMP-14 is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al..
An excitation wavelength of 328nm and an emission wavelength of 393nm are set in an appropriate fluorimeter. The instrument is calibrated with the unquenched peptide Mca-Pro-Leu at a concentration corresponding to between 2 and 10% hydrolysis of the protease substrate. Kinetic reactions are conveniently carried out in a constant volume of 2.5ml.
The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8µM and equilibrated at a temperature of 37°C.
Aliquots of 4 to 8µl of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 minutes.
Activity units per ml enzyme solution are calculated according to the following equation:

Activity (U/ml) = (CMca-Pro-Leu/FMca-Pro-Leu) x (δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg/Venzyme) x Vtotal

CMca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmoles/ml).
FMca-Pro-Leu: Fluorescence of Mca-Pro-Leu at the concentration CMca-Pro-Leu used for fluorimeter calibration.
δFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg: Change in fluorescence during peptide hydrolysis per min.
Vtotal: Volume of peptide hydrolysis reaction (2.5ml).
Venzyme: Volume of added enzyme (0.002 to 0.004ml).
Due to autoproteolytic activity minor bands of activated enzyme may be visible in the preparation.

 

Product Literature References

A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases: C.G. Knight, et al.; FEBS Lett. 296, 263 (1992), Abstract;

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