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CBA003 InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay kit, Fluorogenic

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Synonymes: MMP-2/MMP-9 Activity Assay Kit, Fluorogenic

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CBA003
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Detection Methods
Fluorogenic

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      Description
      OverviewA sensitive fluorogenic assay (Exc. max: ~325 nm; Emi. max.: ~393 nm) for the measurement of gelatinases (MMP-2 and MMP-9). Also useful for the screening of gelatinase inhibitors.
      Catalogue NumberCBA003
      Brand Family Calbiochem®
      SynonymsMMP-2/MMP-9 Activity Assay Kit, Fluorogenic
      Application Data
      Human Pro-MMP-2 (supplied) was activated as outlined in the Reagent Preparation section above and assayed for activity as outlined in the Detailed Protocol, incubating for a total of 3 h.

      Activated MMP-9 (Cat. No. PF140) was assayed without APMA at 37°C for 3 h according to the Detailed Protocol outlined above.

      Serum from 5 different donors was diluted 1:5 in Activation Buffer and HT1080 culture supernatant was diluted 1:3 with Activation Buffer. All samples were incubated at 37°C for 3 h according to the Detailed Protocol outlined above.
      Materials Required but Not Delivered• Pipettors with disposable tips
      • Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set at an excitation wavelength of 320 nm and an emission wavelength of 405 nm
      • Activated MMP-2 (Cat. No. PF023) and/or activated MMP-9 (Cat. No. PF140) (optional positive controls)
      References
      ReferencesLauer-Fields, J.L., et al. 2003. J. Biol. Chem. 278, 18140.
      Chang, C., and Werb, Z. 2001. Trends Cell Biol. 11, S37.
      Kleiner, D.E., and Stetler-Stevenson, W.G. 1999. Cancer Chemother. Pharmacol. 43 (suppl.), S42.
      Cockett, M.I., et al. 1998. Biochem. Soc. Symp. 63, 295.
      Olson, M.W., et al. 1997. J. Biol. Chem. 272, 29975.
      Will, H., et al. 1996. J. Biol. Chem. 271, 17119.
      MacDougall, J.R., et al. 1995. Cancer Metastasis Rev. 14, 351.
      Liotta, L.A., et al. 1990. Semin. Cancer Biol. 1, 99.
      Product Information
      Detection methodFluorogenic
      Form96 Tests
      Format96-well plate
      HS Code3822 19 90
      Kit containsGelatinase Substrate, pro-MMP-2 Positive Control, Inhibitor, APMA, Assay Buffer, 96-Well Plate, Plate Sealer, and a user protocol
      Positive controlMMP-9 (Cat. No. PF140)
      Quality LevelMQ100
      Applications
      Biological Information
      Assay time2 - 6 h
      Sample TypeCell lysate, culture supernatant, tissue extract, plasma, serum, purified enzyme
      Physicochemical Information
      Sensitivity20 ng/ml
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 28-33-36/38-40-43-50/53-60

      Very toxic if swallowed.
      Danger of cumulative effects.
      Irritating to eyes and skin.
      Limited evidence of a carcinogenic effect.
      May cause sensitization by skin contact.
      Very toxic to aquatic organisms; may cause long-term adverse effects in the aquatic environment.
      May impair fertility.
      S PhraseS: 22-26-36/37-57-60-61

      Do not breathe dust.
      In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
      Wear suitable protective clothing and gloves.
      Use appropriate containment to avoid environmental contamination.
      This material and its container must be disposed of as hazardous waste.
      Avoid release to the environment. Refer to special instructions/safety data sheet.
      Product Usage Statements
      Intended useThe Calbiochem® InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay Kit is designed for measuring gelatinase (MMP-2 and MMP-9) activity in a variety of samples. It is most suitable for screening gelatinase inhibitors using purified enzymes. It is not as sensitive as zymography, but it is still sufficient for measuring activity in biological samples (e.g., serum and plasma), culture supernatant, or tissue extracts. The assay utilizes a triple-helical peptide substrate that is highly selective for MMP-2 and MMP-9.
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Multiple Toxicity Values, refer to MSDS
      Hazardous Materials Attention: Due to the nature of the Hazardous Materials in this shipment, additional shipping charges may be applied to your order. Certain sizes may be exempt from the additional hazardous materials shipping charges. Please contact your local sales office for more information regarding these charges.
      Storage -20°C
      Storage ConditionsUpon arrival store the MMP-2 at -70°C and all other contents of the kit at -20°C.
      Protect from Light Protect from light
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit containsGelatinase Substrate, pro-MMP-2 Positive Control, Inhibitor, APMA, Assay Buffer, 96-Well Plate, Plate Sealer, and a user protocol
      Specifications
      Global Trade Item Number
      Référence GTIN
      CBA003-1KIT 04055977220827

      Documentation

      InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay kit, Fluorogenic FDS

      Titre

      Fiche de données de sécurité des matériaux (FDS) 

      InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay kit, Fluorogenic Certificats d'analyse

      TitreNuméro de lot
      CBA003

      Références bibliographiques

      Aperçu de la référence bibliographique
      Lauer-Fields, J.L., et al. 2003. J. Biol. Chem. 278, 18140.
      Chang, C., and Werb, Z. 2001. Trends Cell Biol. 11, S37.
      Kleiner, D.E., and Stetler-Stevenson, W.G. 1999. Cancer Chemother. Pharmacol. 43 (suppl.), S42.
      Cockett, M.I., et al. 1998. Biochem. Soc. Symp. 63, 295.
      Olson, M.W., et al. 1997. J. Biol. Chem. 272, 29975.
      Will, H., et al. 1996. J. Biol. Chem. 271, 17119.
      MacDougall, J.R., et al. 1995. Cancer Metastasis Rev. 14, 351.
      Liotta, L.A., et al. 1990. Semin. Cancer Biol. 1, 99.

      Brochure

      Titre
      Kit SourceBook - 2nd Edition EURO
      Protocole Utilisateur

      Revision08-March-2010 JSW
      SynonymsMMP-2/MMP-9 Activity Assay Kit, Fluorogenic
      Form96 Tests
      Format96-well plate
      Detection methodFluorogenic
      StorageUpon arrival store the MMP-2 at -70°C and all other contents of the kit at -20°C.
      Intended useThe Calbiochem® InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay Kit is designed for measuring gelatinase (MMP-2 and MMP-9) activity in a variety of samples. It is most suitable for screening gelatinase inhibitors using purified enzymes. It is not as sensitive as zymography, but it is still sufficient for measuring activity in biological samples (e.g., serum and plasma), culture supernatant, or tissue extracts. The assay utilizes a triple-helical peptide substrate that is highly selective for MMP-2 and MMP-9.
      BackgroundThe gelatinases, MMP-2 (gelatinase A; 72 kDa) and MMP-9 (gelatinase B; 92 kDa), are two members of the matrix metalloproteinase (MMP) family of enzymes, which are a group of zinc-dependent endopeptidases known to hydrolyze many components of the extracellular matrix, including collagens, elastin, fibronectin, and proteoglycans. All MMPs are produced in a latent form (pro-MMP or zymogen) that requires activation. A unique characteristic of the gelatinases is the ability of the zymogens to form tight non-covalent and stable complexes with tissue inhibitors of metalloproteinases (TIMPs). This proenzyme-inhibitor complex plays a role in zymogen stabilization and activation. Several members of the MMP family have been implicated in tumor cell invasion and angiogenesis as being among the primary proteases overexpressed by tumor cells and responsible for degrading the extracellular matrix (ECM) surrounding the tumor.
      Principles of the assayThe Calbiochem® InnoZyme™ Gelatinase (MMP-2/MMP-9) Activity Assay Kit utilizes a triple-helical, collagen-like, fluorogenic substrate that is highly selective for MMP-2 and MMP-9 [(Gly-Pro-Hyp*)5-Gly-Pro-Lys(Mca)-Gly-Pro-Pro-Gly/Val-Val-Gly-Glu-Lys(Dnp)-Gly-Glu-Gln-(Gly-Pro-Hyp)5-NH2). *Hyp, 4-hydroxy-L-proline; / = cleavage site]. Gelatinases in the sample cleave the substrate, resulting in an increase in fluorescence. The fluorescence increase is measured at an excitation wavelength of 320 nm and an emission wavelength of 405 nm. Similar to native collagen, the substrate is not cleaved by the catalytic domain of MMP-2 or MMP-9 lacking the C-terminal hemopexin domain. The substrate is not cleaved by MMP-1, MMP-3, MMP-13, or MMP-14.
      Materials provided• Gelatinase (MMP-2/MMP-9) Substrate (Kit Component No. JA7760-200UL): 1 amber vial, 200 µl, 750 µM in DMSO
      • Gelatinase (MMP-2/MMP-9) Control (Kit Component No. JA7761-2.5 UG): 1 vial, 2.5 µg, supplied as 0.2 mg/ml solution
      • p-Aminophenylmercuric Acetate (APMA) (Kit Component No. JA7763-500UL): 1 vial, 500 µl, 1 M in DMSO
      • Gelatinase (MMP-2/MMP-9) Inhibitor (Kit Component No. JA7762-50UL): 1 vial, 50 µl, 2 mM MMP-2/MMP-9 Inhibitor II in DMSO
      • Assay Buffer (Kit Component No. JA7764-25ML): 1 bottle, 25 ml
      • 96-Well Plate (Kit Component No. JA7666-1EA): 1 plate, 96-wells, supplied as six 16-well strips (black)
      • Plate Sealer: 2 each
      Materials Required but not provided• Pipettors with disposable tips
      • Fluorescence reader capable of measuring fluorescence in 96-well plates with a filter set at an excitation wavelength of 320 nm and an emission wavelength of 405 nm
      • Activated MMP-2 (Cat. No. PF023) and/or activated MMP-9 (Cat. No. PF140) (optional positive controls)
      PreparationAll samples should be diluted in Activation Buffer just prior to performing the assay. The recommended dilution for serum/plasma is 1:5-1:10; the recommended dilution for culture supernatant is 1:3-1:5.
      Reagent preparationWarm all reagents, except the positive control, to room temperature prior to performing the assay. The Gelatinase (MMP-2/MMP-9) Control should be kept on ice until use. • Activation Buffer: Add 5 µl p-Aminophenylmercuric Acetate (APMA) to a tube containing 5 ml Assay Buffer. If desired, a smaller or larger volume of Activation Buffer can be prepared; the ratio of APMA to Assay Buffer should be 1:1000. Note: the p-Aminophenylmercuric Acetate (APMA) can be stored at –20°C and thawed again for future use. The Activation Buffer should be made fresh for each application and discarded after use. • Substrate Working Solution: Dilute the Gelatinase (MMP-2/MMP-9) Substrate 1:5 with dH2O. For example, to prepare enough reagent for one 16-well strip mix 40 µl Gelatinase (MMP-2/MMP-9) Substrate with 160 µl dH2O. Note: the Gelatinase (MMP-2/MMP-9) Substrate can withstand several freeze/thaw cycles, but it is recommended that it be dispensed into small amounts (~20 µl) for maximum stability. • Inhibitor Working Solution: Dilute the Gelatinase (MMP-2/MMP-9) Inhibitor 1:50 with Activation Buffer. For example, to prepare enough for 5 wells, mix 5 µl Gelatinase (MMP-2/MMP-9) Inhibitor with 245 µl Activation Buffer. • Gelatinase (MMP-2/MMP-9) Control: The Gelatinase (MMP-2/MMP-9) Control must be activated prior to performing the assay. Add 1 µg of Gelatinase (MMP-2/MMP-9) Control to 400 µl Activation Buffer to yield a 2.5 µg/ml solution. Prepare serial dilutions of the reconstituted/activated Gelatinase (MMP-2/MMP-9) Control in Activation Buffer ranging from ~20 to 2000 ng/ml; these dilutions can be used to generate a standard curve for the assay. Note: the reconstituted Gelatinase (MMP-2/MMP-9) Control can be dispensed into smaller aliquots (5 or 10 µl) and stored for several months at -20°C.
      Detailed protocolA. Protocol for Measuring Gelatinase (MMP-2/MM:-9) Activity in Biological Samples

      1. Add 90 µl of each Gelatinase (MMP-2/MMP-9) Standard and sample, diluted in Activation Buffer, to designated wells.
      2. Add 10 µl Substrate Working Solution to each well.
      3. Incubate the wells at 37°C for 2-6 h.
      Note: The Gelatinase (MMP-2/MMP-9) Control is activated in 2-3 h; MMP-9 can be fully activated in ~4-6 h, but may take up to 18 h, depending on the nature of the pro-MMP-9 preparation. The presence of tissue inhibitors of metalloproteinase (TIMPs) will require that the sample be incubated for an extended period to achieve complete activation.
      4. Measure the fluorescence using a fluorescence plate reader set at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.

      B. Protocol for Screening Gelatinase Inhibitors

      1. Add 45 µl Inhibitor Working Solution to designated wells. If screening test inhibitors, dilute to the desired concentration(s) in Activation Buffer and add 45 µl test inhibitor(s)to designated wells.
      2. Add 45 µl activated MMP-2 or MMP-9 (only MMP-2 is provided) diluted in Activation Buffer to each well.
      Note: if using a preparation of MMP-2 or MMP-9 that is already activated it is not necessary to perform the activation step as outlined in the Reagent Preparation section above; the enzyme can then be diluted in Assay Buffer without APMA just prior to performing the assay.
      3. Incubate at room temperature for 5-15 min.
      4. Add 10 µl Substrate Working Solution to each well.
      5. Incubate at 37°C for 2-3 h.
      6. Measure the fluorescence using a fluorescence reader set at an excitation wavelength of 320 nm and an emission wavelength of 405 nm.
      Example data

      Figure 1: Activity of Human Pro-MMP-2

      Human Pro-MMP-2 (supplied) was activated as outlined in the Reagent Preparation section above and assayed for activity as outlined in the Detailed Protocol, incubating for a total of 3 h.

      Figure 2: Activity of MMP-9

      Activated MMP-9 (Cat. No. PF140) was assayed without APMA at 37°C for 3 h according to the Detailed Protocol outlined above.

      Figure 3: Inhibition of MMP-2 and MMP-9 Activity

      MMP-2 and MMP-9 were incubated in the presence or absence of MMP-2/MMP-9 Inhibitor II (Cat. No. 444249) and activity assayed as outlined in protocol B of the Detailed Protocol above.

      Figure 4: Gelatinase Activity in Biological Samples

      Serum from 5 different donors was diluted 1:5 in Activation Buffer and HT1080 culture supernatant was diluted 1:3 with Activation Buffer. All samples were incubated at 37°C for 3 h according to the Detailed Protocol outlined above.
      Sensitivity20 ng/ml
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      InnoZyme™ and InterActive Pathways™ are trademarks of EMD Chemicals, Inc.