ALEXIS Biochemicals: MMP-14 Prodomain (Catalytic Domain) (human) (recombinant) (His)

ALX-201-099

Revised 08-Jan-08

MMP-14 Prodomain (Catalytic Domain) (human) (recombinant) (His)
PRODUCT LINE	Cytoskeleton
PRODUCT CATEGORY	MMPs

Ordering Information

Product Numbers:	Format:	Size:	Unit Price:	Quantity:	Add To Cart
ALX-201-099-C010		10 µg	560.00 USD

Product Specification

MW:	~31kDa
EC:	3.4.24.-
SOURCE/HOST:	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).
CONCENTRATION:	0.2mg/ml
PURITY:	≥90% (SDS-PAGE)
FORMULATION:	Liquid. In 50mM TRIS-HCl, pH 7.5, containing 150mM NaCl and 5mMCaCl₂.
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.
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.

Product Description

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.

Product Specific Literature References

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

General Information

BACKGROUND/TECHNICAL INFORMATION

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-NH₂in 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. [1].
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) = (C_Mca-Pro-Leu/F_Mca-Pro-Leu) x (ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}/V_enzyme) x V_total

C_Mca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmoles/ml).
F_Mca-Pro-Leu: Fluorescence of Mca-Pro-Leu at the concentration C_Mca-Pro-Leuused for fluorimeter calibration.
ΔF_{Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg}: Change in fluorescence during peptide hydrolysis per min.
V_total: Volume of peptide hydrolysis reaction (2.5ml).
V_enzyme: Volume of added enzyme (0.002 to 0.004ml).

Due to autoproteolytic activity minor bands of activated enzyme may be visible in the preparation.

Swiss-Prot link P50281: MMP-14 (human) (precursor)
AfCS Signalling Gateway link A001466: MMP-14 (mouse)

Further Categories Containing This Product:

Enzymes • Recombinant Proteins / Fusion Proteins

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