Progelatinase B Protein

Details for Product No. ABIN368651, Supplier: Log in to see
Protein Name
Origin
Human
Source
Human
Protein Type
Native
Application
Screening Assay (ScA), Standard (STD)
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Specificity Gelatinase B Human progelatinase B consists of 668 amino acids.
Cross-Reactivity (Details) No cross reactivity:
Characteristics Progelatinase B monomer is isolated from human blood. The preparation is free from gelatinase B dimer and from complexes of gelatinase B with TIMP-1 or lipocalin.
Purity > 95 %
Background Synonyms: Progelatinase B Monomer, 92- kDa Type IV Collagenase Monomer
Gene Name: matrix metallopeptidase 9
Gene: MMP9
Molecular Weight 92 kDa
Gene ID 4318
UniProt P14780
Application Notes Degradation of extracellular Matrix, Screening and evaluation of MMP inhibitors, Antigen standard
Reagent Preparation

Preparation and stability of solutions: Activation buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2. Trypsin solution: 0.50 mg TPCK-trypsin / mL activation buffer. The solution is stored in aliquots at -20 °C. Aprotinin solution: 1 mg aprotinin / mL activation buffer. The solution is stored at - 20 °C. Peptide hydrolysis buffer: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl 2 , 0.025 % Brij 35. The solution is stable for several weeks at 4 °C. Stock solution of peptide substrate: 100 µM solution of Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg in 20 % dimethylsulfoxide. The solution is stored at -20 °C. Stock solution of unquenched peptide: 10 µM solution of (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH 2 (Mca-Pro-Leu) in 20 % dimethylsulfoxide. The solution is stored at -20 °C. 4.2 Activation: Aliquots of 10 µL progelatinase B monomer are mixed with 20 µL trypsin solution and activation buffer in a total volume of 100 ul. The mixture is incubated for 20 min at 37 °C. Thereafter trypsin is inhibited by addition of 10 µL aprotinin solution.

Assay Procedure

The activity of gelatinase B is measured fluorimetrically with a synthetic internally quenched fluorescent substrate according to Knight et al. [15]. An excitation wavelength of 328 nm and an emission wavelength of 393 nm 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 2.5 mL . The substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg is diluted in peptide hydrolysis buffer to a concentration of 0.8 µM and equilibrated at a temperature of 37 °C. Aliquots of 5 µL to 10 µL of the activation mixture are than added and the increase in fluorescence is recorded over a time interval between 2 and 12 min. Activity units per mL enzyme solution are calculated according to the following equation: c Mca-Pro-Leu deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg Activity U/mL = ?????.. V total F Mca-Pro-Leu v enzyme c Mca-Pro-Leu : Concentration of Mca-Pro-Leu used for calibration of the fluorimeter ( umoles/mL) F Mca-Pro-Leu : Fluorescence of Mca-Pro-Leu at the concentration c Mca-Pro-Leu used for fluorimeter calibration deltaF Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg : Change in fluorescence during peptide hydrolysis per min V : Volume of peptide hydrolysis reaction (2.5 mL ) v : Volume of added enzyme (0.005 mL to 0.010 mL ) .

Restrictions For Research Use only
Format Liquid
Buffer 50 mM Tris-HCl, pH 7.0, 200 mM NaCl, 5 mM CaCl2, 1 μM ZnCl2, 0.05 % Brij-35, 0.05 % Sodium azide
Preservative Sodium azide
Precaution of Use This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Storage -80 °C
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 image for Progelatinase B protein (ABIN368651) Progelatinase B protein
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