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PF038 MMP-9, Proenzyme, Human, Recombinant, CHO Cells

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

Synonyms: Gelatinase B, 92 kDa Gelatinase, Matrix Metalloproteinase 9, Type IV Collagenase

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PF038
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PF038-10UG
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      Description
      OverviewRecombinant, human pro-MMP-9 expressed in CHO cells. A portion (less than 10%) of the enzyme may be seen as a naturally-occurring dimer. Dimerization does not interfere with activation. May contain up to 10% TIMP proteins. During storage, a small portion (less than 10%) of the enzyme may also become activated. Requires activation prior to use. A simple activation protocol is included.
      Catalogue NumberPF038
      Brand Family Calbiochem®
      SynonymsGelatinase B, 92 kDa Gelatinase, Matrix Metalloproteinase 9, Type IV Collagenase
      References
      ReferencesParsons, S.L., et al. 1997. Br. J. Surg. 84, 160.
      Backstrom, J.R., et al. 1996. J. Neuro. 16, 7910.
      Lim, G.P., et al. 1996. J Neurochem. 67, 251.
      Sang, Q.X., et al. 1995. Biochim. Biophys. Acta. 1251, 99.
      Kenagy, R.D. and Clowes, A.W. 1994. in Inhibition of Matrix Metalloproteinases: Therapeutic Potential. Greenwald, R.A. and Golub L.M., Eds.: 462-465.
      Zempo, N., et al. 1994. J. Vasc. Surg. 20, 209.
      Birkedal-Hansen, H. 1993. J. Periodontol. 64, 474.
      Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
      Delaisse, J-M. and Vaes, G. 1992. in Biology and Physiology of the Osteoclast. B.R. Rifkin & C.V. Gay, Eds.: 290-314.
      Jeffrey, J.J. 1992. in Wound Healing: Biochemical and Clinical Aspects. R.F. Diegelmann and W.J. Lindblad, Eds.: 177-194.
      Jeffrey, J.J. 1991. Semin. Perinatol. 15, 118.
      Liotta, L.A., et al. 1991. Cell 64, 327.
      Harris, E. 1990. N. Engl. J. Med. 322, 1277.
      Product Information
      Activity>1,300 pmoles/min/µg
      Unit of DefinitionSpecific activity is determined using 10 μM (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH₂ (excitation 320 nm, emission 405 nm), and 20 ng enzyme in 100 μl of 50 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 150 mM NaCl, and 0.05% BRIJ<sup>®</sup>-35 Detergent at room temperature.
      EC number3.4.24.35
      FormLiquid
      FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 10 mM CaCl₂, 0.05% BRIJ®-35 Detergent, pH 7.5.
      PreservativeNone
      Quality LevelMQ100
      Applications
      Application ReferencesZymography Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259. Kleiner, D.E. and Stetler-Stevenson W.G. 1994. Anal. Biochem. 218, 325. Heussen, C. and Dowdle, E.B. 1980. Anal. Biochem. 102, 196. Substrate Cleavage Assay Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259.
      Key Applications Immunoblotting (Western Blotting)
      Substrate Cleavage Assay
      Zymography
      Application NotesImmunoblotting (1 µg protein/lane)
      Substrate Cleavage Assay (1 µg protein/lane, see application references)
      Zymography (1 µg protein/lane, see application references)
      Application CommentsMMP-9 Proenzyme can be measured by its ability to degrade gelatin in a zymogram. 0.5 ng of enzyme is sufficient to visualize degraded gelatin with coomassie blue stain. The specific activity as measured with 10 µM (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2 (excitation 320 nm, emission 405 nm), and 20 ng enzyme in 100 µl of 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35 at room temperature, is >1,300 pmoles/min/µg. To activate MMP-9 proenzyme, prepare p-aminophenylmercuric acetate (APMA) concentrate in dimethylsulfoxide (DMSO). Add APMA to MMP-9 proenzyme to give a final APMA concentration of 1 mM. Incubate at 37°C for 16 to 24 h.
      Biological Information
      Purity>90% by SDS-PAGE
      Concentration Label Please refer to vial label for lot-specific concentration
      Physicochemical Information
      Dimensions
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Dry Ice Only
      Toxicity Standard Handling
      Storage ≤ -70°C
      Avoid freeze/thaw Avoid freeze/thaw
      Do not freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Packaging Information
      Transport Information
      Supplemental Information
      Specifications
      Global Trade Item Number
      Catalogue Number GTIN
      PF038-10UG 04055977207408

      Documentation

      MMP-9, Proenzyme, Human, Recombinant, CHO Cells SDS

      Title

      Safety Data Sheet (SDS) 

      MMP-9, Proenzyme, Human, Recombinant, CHO Cells Certificates of Analysis

      TitleLot Number
      PF038

      References

      Reference overview
      Parsons, S.L., et al. 1997. Br. J. Surg. 84, 160.
      Backstrom, J.R., et al. 1996. J. Neuro. 16, 7910.
      Lim, G.P., et al. 1996. J Neurochem. 67, 251.
      Sang, Q.X., et al. 1995. Biochim. Biophys. Acta. 1251, 99.
      Kenagy, R.D. and Clowes, A.W. 1994. in Inhibition of Matrix Metalloproteinases: Therapeutic Potential. Greenwald, R.A. and Golub L.M., Eds.: 462-465.
      Zempo, N., et al. 1994. J. Vasc. Surg. 20, 209.
      Birkedal-Hansen, H. 1993. J. Periodontol. 64, 474.
      Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
      Delaisse, J-M. and Vaes, G. 1992. in Biology and Physiology of the Osteoclast. B.R. Rifkin & C.V. Gay, Eds.: 290-314.
      Jeffrey, J.J. 1992. in Wound Healing: Biochemical and Clinical Aspects. R.F. Diegelmann and W.J. Lindblad, Eds.: 177-194.
      Jeffrey, J.J. 1991. Semin. Perinatol. 15, 118.
      Liotta, L.A., et al. 1991. Cell 64, 327.
      Harris, E. 1990. N. Engl. J. Med. 322, 1277.
      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.

      Revision23-April-2010 JSW
      SynonymsGelatinase B, 92 kDa Gelatinase, Matrix Metalloproteinase 9, Type IV Collagenase
      ApplicationImmunoblotting (1 µg protein/lane)
      Substrate Cleavage Assay (1 µg protein/lane, see application references)
      Zymography (1 µg protein/lane, see application references)
      DescriptionRecombinant, human pro-MMP-9 expressed in CHO cells. The calculated molecular weight is ~77 kDa, but the apparent molecular weight is ~92 kDa by SDS-PAGE. Useful for immunoblotting, substrate cleavage assays, and zymography. MMP-9 Proenzyme can be measured by its ability to degrade gelatin in a zymogram. 0.5 ng of enzyme is sufficient to visualize degraded gelatin with coomassie blue stain. Matrix metalloproteinases are members of a unique family of proteolytic enzymes that have a zinc ion at their active sites and can degrade collagens, elastin and other components of the extracellular matrix (ECM). These enzymes are present in normal healthy individuals and have been shown to have an important role in processes such as wound healing, pregnancy, and bone resorption. However, overexpression and activation of MMPs have been linked with a range of pathological processes and disease states involved in the breakdown and remodeling of the ECM. Such diseases include tumor invasion and metastasis, rheumatoid arthritis, periodontal disease and vascular processes such as angiogenesis, intimal hyperplasia, atherosclerosis and aneurysms. Recently, MMPs have been linked to neurodegenerative diseases such as Alzheimer’s, and amyotrophic lateral sclerosis (ALS). Natural inhibitors of MMPs, tissue inhibitor of matrix metalloproteinases (TIMPs) exist and synthetic inhibitors have been developed which offer hope of new treatment options for these diseases. Regulation of MMP activity can occur at the level of gene expression, including transcription and translation, level of activation, or at the level of inhibition by TIMPs. Thus, perturbations at any of these points can theoretically lead to alterations in ECM turnover. Expression is under tight control by pro- and anti-inflammatory cytokines and/or growth factors and, once produced the enzymes are usually secreted as inactive zymograms. Upon activation (removal of the inhibitory propeptide region of the molecules) MMPs are subject to control by locally produced TIMPs. All MMPs can be activated in vitro with organomercurial compounds (e.g., 4-aminophenylmercuric acetate), but the agents responsible for the physiological activation of all MMPs have not been clearly defined. Numerous studies indicate that members of the MMP family have the ability to activate one another. The activation of the MMPs in vivo is likely to be a critical step in terms of their biological behavior, because it is this activation that will tip the balance in favor of ECM degradation. The hallmark of diseases involving MMPs appear to be stoichiometric imbalance between active MMPs and TIMPs, leading to excessive tissue disruption and often degradation. Determination of the mechanisms that control this imbalance may open up some important therapeutic options of specific enzyme inhibitors.
      BackgroundMatrix metalloproteinases are members of a unique family of proteolytic enzymes that have a zinc ion at their active sites and can degrade collagens, elastin and other components of the extracellular matrix (ECM). These enzymes are present in normal healthy individuals and have been shown to have an important role in processes such as wound healing, pregnancy, and bone resorption. However, overexpression and activation of MMPs have been linked with a range of pathological processes and disease states involved in the breakdown and remodeling of the ECM. Such diseases include tumor invasion and metastasis, rheumatoid arthritis, periodontal disease and vascular processes such as angiogenesis, intimal hyperplasia, atherosclerosis and aneurysms. Recently, MMPs have been linked to neurodegenerative diseases such as Alzheimer’s, and amyotrophic lateral sclerosis (ALS). Natural inhibitors of MMPs, tissue inhibitor of matrix metalloproteinases (TIMPs) exist and synthetic inhibitors have been developed which offer hope of new treatment options for these diseases. Regulation of MMP activity can occur at the level of gene expression, including transcription and translation, level of activation, or at the level of inhibition by TIMPs. Thus, perturbations at any of these points can theoretically lead to alterations in ECM turnover. Expression is under tight control by pro- and anti-inflammatory cytokines and/or growth factors and, once produced the enzymes are usually secreted as inactive zymograms. Upon activation (removal of the inhibitory propeptide region of the molecules) MMPs are subject to control by locally produced TIMPs. All MMPs can be activated in vitro with organomercurial compounds (e.g., 4-aminophenylmercuric acetate), but the agents responsible for the physiological activation of all MMPs have not been clearly defined. Numerous studies indicate that members of the MMP family have the ability to activate one another. The activation of the MMPs in vivo is likely to be a critical step in terms of their biological behavior, because it is this activation that will tip the balance in favor of ECM degradation. The hallmark of diseases involving MMPs appear to be stoichiometric imbalance between active MMPs and TIMPs, leading to excessive tissue disruption and often degradation. Determination of the mechanisms that control this imbalance may open up some important therapeutic options of specific enzyme inhibitors.
      FormLiquid
      FormulationIn 150 mM NaCl, 50 mM Tris-HCl, 10 mM CaCl₂, 0.05% BRIJ®-35 Detergent, pH 7.5.
      Concentration Label Please refer to vial label for lot-specific concentration
      Recommended reaction conditions

      Organomercurial Activation Protocol This protocol is provided only as a general guide. Researchers should standardize this assay for their own specific needs and should consult published literature. The following protocol is from Stricklin, et al., which describes the use of p-aminophenylmercuric acetate (APMA) to activate pro-MMP. This protocol is also adaptable to other types of organomercurals, such as p-(hydroxymercuric) benzoate (PHMB), phenylmercuric chloride (PMC), or mersalyl. 1. Prepare a 10-50 mM stock solution of APMA (or other organomercurial compound) in 0.1 M NaOH just prior to use. Although not absolutely necessary, the stock solution may be adjusted to pH 11 with 5 N HCl (see Marcy, A.I., et al.). 2. To initiate the activation mix the proenzyme solution with the APMA solution at a 10:1 volume ratio (MMP:APMA). If a higher concentration of APMA is desired, increase the concentration of the stock solution. Do not exceed the 10:1 ratio, as this could result in significant changes in pH. 3. Incubate the mixture at 37°C for 2-3 h. It is recommended that an analytical run be conducted first to determine the optimal incubation time. For example, a small-scale experiment with a fixed concentration of pro-MMP and organomercurial would be incubated as described above. Remove aliquots of the sample at various time points during the incubation. Stop the reaction by the addition of SDS-PAGE sample buffer (e.g., 10 µl 2X sample buffer to 10 µl aliquot) and heat the samples to 95°C. The progress of activation can be monitored qualitatively by analyzing the aliquots on a 12% SDS-PAGE gel. 4. The activated MMP can be used without removing the APMA from the mixture. Please refer to Marcy, A.I., et al. for removal of organomercurials by gel filtration.
      EC number3.4.24.35
      Purity>90% by SDS-PAGE
      Activity>1,300 pmoles/min/µg
      Unit definitionSpecific activity is determined using 10 μM (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH₂ (excitation 320 nm, emission 405 nm), and 20 ng enzyme in 100 μl of 50 mM Tris-HCl, pH 7.5, 10 mM CaCl₂, 150 mM NaCl, and 0.05% BRIJ®-35 Detergent at room temperature.
      PreservativeNone
      CommentsMMP-9 Proenzyme can be measured by its ability to degrade gelatin in a zymogram. 0.5 ng of enzyme is sufficient to visualize degraded gelatin with coomassie blue stain. The specific activity as measured with 10 µM (7-methoxycoumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2 (excitation 320 nm, emission 405 nm), and 20 ng enzyme in 100 µl of 50 mM Tris-HCl, pH 7.5, 10 mM CaCl2, 150 mM NaCl, and 0.05% Brij-35 at room temperature, is >1,300 pmoles/min/µg. To activate MMP-9 proenzyme, prepare p-aminophenylmercuric acetate (APMA) concentrate in dimethylsulfoxide (DMSO). Add APMA to MMP-9 proenzyme to give a final APMA concentration of 1 mM. Incubate at 37°C for 16 to 24 h.
      Storage Avoid freeze/thaw
      ≤ -70°C
      Do Not Freeze Ok to freeze
      Special InstructionsFollowing initial thaw, aliquot and freeze (-70°C).
      Toxicity Standard Handling
      ReferencesParsons, S.L., et al. 1997. Br. J. Surg. 84, 160.
      Backstrom, J.R., et al. 1996. J. Neuro. 16, 7910.
      Lim, G.P., et al. 1996. J Neurochem. 67, 251.
      Sang, Q.X., et al. 1995. Biochim. Biophys. Acta. 1251, 99.
      Kenagy, R.D. and Clowes, A.W. 1994. in Inhibition of Matrix Metalloproteinases: Therapeutic Potential. Greenwald, R.A. and Golub L.M., Eds.: 462-465.
      Zempo, N., et al. 1994. J. Vasc. Surg. 20, 209.
      Birkedal-Hansen, H. 1993. J. Periodontol. 64, 474.
      Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
      Delaisse, J-M. and Vaes, G. 1992. in Biology and Physiology of the Osteoclast. B.R. Rifkin & C.V. Gay, Eds.: 290-314.
      Jeffrey, J.J. 1992. in Wound Healing: Biochemical and Clinical Aspects. R.F. Diegelmann and W.J. Lindblad, Eds.: 177-194.
      Jeffrey, J.J. 1991. Semin. Perinatol. 15, 118.
      Liotta, L.A., et al. 1991. Cell 64, 327.
      Harris, E. 1990. N. Engl. J. Med. 322, 1277.
      Application referencesZymography Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259. Kleiner, D.E. and Stetler-Stevenson W.G. 1994. Anal. Biochem. 218, 325. Heussen, C. and Dowdle, E.B. 1980. Anal. Biochem. 102, 196. Substrate Cleavage Assay Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259.