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

CBA003
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      Descripción

      Replacement Information

      Tabla espec. clave

      Detection Methods
      Fluorogenic

      Precios y disponibilidad

      Número de referenciaDisponiblidad Embalaje Cant./Env. Precio Cantidad
      US1CBA003-1KIT
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          1 kit
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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
          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)
          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

          Documentation

          Ficha datos de seguridad (MSDS)

          Título

          Ficha técnica de seguridad del material (MSDS) 

          Certificados de análisis

          CargoNúmero de lote
          CBA003

          Referencias bibliográficas

          Visión general referencias
          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.
          Protocolo de usuario

          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.