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48-602MAG
Buffer Detection Kit for Magnetic Beads
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475935
Sigma-AldrichMT1-MMP, Catalytic Domain, Human, Recombinant, E. coli
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Description
Overview
Recombinant, human pro-MT1-MMP expressed in E. coli and activated to yield the active catalytic domain (amino acids 89-265). Degrades fibrillar collagen Types I, II, and III, as well as other extracellular matrix proteins, and activates MMP-2 and MMP-13 resulting in enhanced proteolysis of the matrix for cell migration and proliferation.
Note: 1 mU = 1 milliunit.
Catalogue Number
475935
Brand Family
Calbiochem®
Synonyms
MMP-14 Catalytic Domain
References
References
Amano, T., et al. 2005. Cell Res.15, 150. Knauper, V., et al. 1996. J. Biol. Chem. 271, 17124. Pei, D., and Weiss, S.J. 1996. J. Biol. Chem. 271, 9135. Sato, H., et al. 1994. Nature370, 61. Knight, C.G., et al. 1992. REBS Lett.296, 263.
Product Information
Unit of Definition
One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (<a href ="/Products/ProductDisplay.asp→catNO=03-32-5032">Cat. No. 03-32-5032</a>) per min at 37°C, pH 7.5.
Form
Liquid
Formulation
In 150 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl₂, pH 7.5.
MT1-MMP, Catalytic Domain, Human, Recombinant, E. coli Certificates of Analysis
Title
Lot Number
475935
References
Reference overview
Amano, T., et al. 2005. Cell Res.15, 150. Knauper, V., et al. 1996. J. Biol. Chem. 271, 17124. Pei, D., and Weiss, S.J. 1996. J. Biol. Chem. 271, 9135. Sato, H., et al. 1994. Nature370, 61. Knight, C.G., et al. 1992. REBS Lett.296, 263.
Data Sheet
Note that this data sheet is not lot-specific and is representative of the current specifications for this product. Please consult the vial label and the certificate of analysis for information on specific lots. Also note that shipping conditions may differ from storage conditions.
Revision
17-September-2007 RFH
Synonyms
MMP-14 Catalytic Domain
Description
Recombinant, human pro-MT1-MMP expressed in E. coli and activated to yield the active catalytic domain (amino acids 89-265). The catalytic domain is comprised of amino acid residues Tyr89 to Gly265 of mature human MT1-MMP. Hydrolyzes fibrillar collagens I, II, and III, as well as other extracellular matrix proteins, and activates MMP-2 and MMP-13, resulting in enhanced proteolysis of the matrix for cell migration and proliferation.
Form
Liquid
Formulation
In 150 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl₂, pH 7.5.
Concentration Label
Please refer to vial label for lot-specific concentration
Recommended reaction conditions
Activity Assay Reagent Preparation and Required EquipmentPeptide Hydrolysis Buffer: 150 mM NaCl, 50 mM Tris-HCl, 5 mM CaCl2, 0.025 % Brij® 35 detergent, pH 7.5. Store at 4°C; stable for several weeks.
Peptide Substrate Stock: 100 µM Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg (Cat. No. 05-32-5032) in 20% DMSO. Store at -20°C.
Peptide Standard: 10 µM (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-NH2 (Mca-Pro-Leu) (Cat. No. 05-32-5033) in 20% DMSO. Store at -20°C.
Fluorimeter: Excitation wavelength: ~328 nm; emission wavelength: ~393 nm
ProtocolNote: This protocol is provided only as a guide; assay conditions should be optimized for individual experiments. The protocol is based on the following reference:
Knight, C.G., et al. 1992. FEBS Lett.296, 263.
1. Calibrate the fluorimeter using the Peptide Standard (Mca-Pro-Leu) at a concentration that corresponds to 2–10% hydrolysis of the Peptide Substrate.
2. Kinetic Dilute the Peptide Substrate Stock to 8 µM in Peptide Hydrolysis Buffer and equilibrate to 37°C.
3. Add 2–4 µl MT1-MMP and record the increase in fluorescence at desired intervals from 2–12 min. 4. Calculate activity in units per ml enzyme using the following equation:
Activity (U/ml) = cMca-Pro-Leu/FMca-Pro-Leu x ΔFMca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg/venzyme x VtotalcMca-Pro-Leu: Concentration of Mca-Pro-Leu used for calibration of the fluorimeter (µmol/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
V: Volume of peptide hydrolysis reaction (2.5 ml)
v: Volume of added enzyme (2–4 µl)
Purity
≥90% by SDS-PAGE
Specific activity
≥140 mU/mg protein
Unit definition
One unit is defined as the amount of enzyme that will hydrolyze 1.0 µmol MCA-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH₂ (Cat. No. 03-32-5032) per min at 37°C, pH 7.5.
Storage
Avoid freeze/thaw ≤ -70°C
Do Not Freeze
Ok to freeze
Special Instructions
Following initial thaw, aliquot and freeze (-70°C).
Toxicity
Standard Handling
References
Amano, T., et al. 2005. Cell Res.15, 150. Knauper, V., et al. 1996. J. Biol. Chem. 271, 17124. Pei, D., and Weiss, S.J. 1996. J. Biol. Chem. 271, 9135. Sato, H., et al. 1994. Nature370, 61. Knight, C.G., et al. 1992. REBS Lett.296, 263.
