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PF063 MMP-3, Proenzyme, Human, Recombinant

MMP-3, Proenzyme, Human, Recombinant: Ficha de datos de seguridad (MSDS o SDS), certificado de análisis y de calidad (CoA y CoQ), expedientes, folletos y otros documentos disponibles.
PF063
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Descripción

Replacement Information

Products

Número de referenciaEmbalaje Cant./Env.
PF063-10UG Ampolla de plást. 10 μg
Description
OverviewRecombinant, human pro-MMP-3 purified from cell culture supernatant. May be used as a positive control or standard for zymographic analysis, or substrate assay. Requires activation for immunoblotting, prior to use. M.W. 57000/58000.
Catalogue NumberPF063
Brand Family Calbiochem®
References
ReferencesSole, S., et al. 2004. J. Neuropathol. Exp. Neurol. 63, 338.
Galazka, G., et al. 1996. Biochem. 35, 11221.
Cottam, D.W. and Rees, R.C. 1993. Intl. J. Oncol. 2, 861.
Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Woessner, J.F. 1991. FASEB J. 5, 2145.
Liotta, L.A. and Stetler-Stevenson, W.G. 1990. In Seminars in Cancer Biology, ed. M.M. Gottesman. Vol. 1, 99.

Zymography References
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 References
Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Product Information
ActivityThe activity of proenzyme MMP 3 was measured by substrate cleavage assay using 0.5 mM thiopeptiolide (Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-Oet) as a substrate. The activity was also assessed by degradation of a peptide substrate (DNP-PYAYWMR) using activated MMP-3 as measured by HPLC.
EC number3.4.24.17
FormLyophilized
FormulationLyophilized from 100 mM NaCl, 50 mM HEPES, pH 7.3.
Quality LevelMQ100
Applications
Application ReferencesZYMOGRAPHY REFERENCES 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 REFERENCE Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259. Netzel-Arnett S, et al. 1991. Anal. Biochem. 195, 86.
Key Applications Immunoblotting (Western Blotting)
Substrate Cleavage Assay
Zymography
Application NotesImmunoblotting (see comments)
Substrate Cleavage Assay (see comments)
Zymography (see comments)
Application CommentsProenzyme MMP-3 may be used as a positive control or standard for immunoblotting, zymographic analysis, or substrate cleavage assays. 0.5 μg/lane was used for SDS-PAGE and immunoblotting. For zymography with casein 1μg/lane of Proenzyme MMM-3 or activated MMP-3 was used. Proenzyme MMP-3 can be activated in vitro by incubation in 50 mM Tris, pH 7.5, containing 0.05% Triton-X-100, 5 mM CaCl2 and 1 mM 4 aminophenyl mercuric acetate (APMA) for 2-4 hours at 37°C. To dissolve APMA, make a 10 mM stock solution in 0.05 M NaOH. Approximately 90% of proenzyme MMP-3 is activated with a 4 hour incubation at 37°C using 1 mM APMA.
Biological Information
Purity≥95% by SDS-PAGE
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 reconstitution, aliquot into siliconized vials and freeze (-70°C).
Packaging Information
Transport Information
Supplemental Information
Specifications
Global Trade Item Number
Número de referencia GTIN
PF063-10UG 04055977207446

Documentation

MMP-3, Proenzyme, Human, Recombinant Ficha datos de seguridad (MSDS)

Título

Ficha técnica de seguridad del material (MSDS) 

MMP-3, Proenzyme, Human, Recombinant Certificados de análisis

CargoNúmero de lote
PF063

Referencias bibliográficas

Visión general referencias
Sole, S., et al. 2004. J. Neuropathol. Exp. Neurol. 63, 338.
Galazka, G., et al. 1996. Biochem. 35, 11221.
Cottam, D.W. and Rees, R.C. 1993. Intl. J. Oncol. 2, 861.
Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Woessner, J.F. 1991. FASEB J. 5, 2145.
Liotta, L.A. and Stetler-Stevenson, W.G. 1990. In Seminars in Cancer Biology, ed. M.M. Gottesman. Vol. 1, 99.

Zymography References
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 References
Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Ficha técnica

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.

Revision14-May-2010 JSW
ApplicationImmunoblotting (see comments)
Substrate Cleavage Assay (see comments)
Zymography (see comments)
DescriptionRecombinant, human pro-MMP-3 purified from cell culture supernatant. May be used as a positive control or standard for zymographic analysis, or substrate assay. Requires activation for immunoblotting, prior to use. M.W. 57000/58000.

Matrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the degradation of extracellular matrix components such as collagen, laminin and proteoglycans. In addition to sequence homology, all MMPs share the following characteristics: the catalytic mechanism is dependent upon a zinc ion at the active center, they cleave one or more extracellular matrix components, they are secreted as zymogens which are activated by removal of an approximately 10 kDa segment from the N terminus and they are inhibited by tissue inhibitor of metalloproteinases (TIMP). These enzymes are involved in normal physiological processes such as embryogenesis and tissue remodeling and may play an important role in angiogenesis, arthritis, periodontitis, and metastasis. Matrix metalloproteinase-3 (MMP-3) also known as stromelysin-1 and transin (EC 3.4.24.17) cleaves a number of substrates including cartilage proteoglycan, collagen types II, III, IV, V and IX, fibronectin, laminin, and can activate MMP 1. MMP-3 is secreted as ~57 and ~59 kDa proenzymes and can be activated in vitro by organomercurials (e.g., 4 aminophenylmercuric acetate, APMA) and in vivo by proteases via intermediate forms to a 45 kDa active MMP 3 enzyme. Further autolysis to a ~28 kDa form can also occur. MMP-3 is thought to play an important role in pathophysiological degradation processes associated with conditions such as rheumatoid arthritis and cancer cell invasion.
BackgroundMatrix metalloproteinases (MMPs) are a family of enzymes that are responsible for the degradation of extracellular matrix components such as collagen, laminin and proteoglycans. In addition to sequence homology, all MMPs share the following characteristics: the catalytic mechanism is dependent upon a zinc ion at the active center, they cleave one or more extracellular matrix components, they are secreted as zymogens which are activated by removal of an approximately 10 kDa segment from the N terminus and they are inhibited by tissue inhibitor of metalloproteinases (TIMP). These enzymes are involved in normal physiological processes such as embryogenesis and tissue remodeling and may play an important role in angiogenesis, arthritis, periodontitis, and metastasis. Matrix metalloproteinase-3 (MMP-3) also known as stromelysin-1 and transin (EC 3.4.24.17) cleaves a number of substrates including cartilage proteoglycan, collagen types II, III, IV, V and IX, fibronectin, laminin, and can activate MMP 1. MMP-3 is secreted as 57 and 59 kDa proenzymes and can be activated in vitro by organomercurials (e.g., 4 aminophenylmercuric acetate, APMA) and in vivo by proteases via intermediate forms to a 45 kDa active MMP 3 enzyme. Further autolysis to a 28 kDa form can also occur. MMP-3 is thought to play an important role in pathophysiological degradation processes associated with conditions such as rheumatoid arthritis and cancer cell invasion.
FormLyophilized
FormulationLyophilized from 100 mM NaCl, 50 mM HEPES, pH 7.3.
Recommended reaction conditionsProenzyme MMP-3 may be used as a positive control or standard for immunoblotting, zymographic analysis, or substrate cleavage assays. 0.5 µg/lane was used for SDS-PAGE and immunoblotting. For zymography with casein 1 µg/lane of Proenzyme MMP-3 or activated MMP-3 was used. Proenzyme MMP-3 can be activated in vitro by incubation in 50 mM Tris, pH 7.5, containing 0.05% Triton®-X-100 detergent, 5 mM CaCl2 and 1 mM 4 aminophenyl mercuric acetate (APMA) for 2-3 h at 37°C. To dissolve APMA, make a 10 mM stock solution in 0.05 M NaOH. Approximately 90% of proenzyme MMP-3 is activated with a 2-3 h incubation at 37°C using 1 mM APMA.

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 number3.4.24.17
Purity≥95% by SDS-PAGE
ActivityThe activity of proenzyme MMP 3 was measured by substrate cleavage assay using 0.5 mM thiopeptiolide (Ac-Pro-Leu-Gly-S-Leu-Leu-Gly-Oet) as a substrate. The activity was also assessed by degradation of a peptide substrate (DNP-PYAYWMR) using activated MMP-3 as measured by HPLC.
SolubilityH₂O
CommentsProenzyme MMP-3 may be used as a positive control or standard for immunoblotting, zymographic analysis, or substrate cleavage assays. 0.5 μg/lane was used for SDS-PAGE and immunoblotting. For zymography with casein 1μg/lane of Proenzyme MMM-3 or activated MMP-3 was used. Proenzyme MMP-3 can be activated in vitro by incubation in 50 mM Tris, pH 7.5, containing 0.05% Triton-X-100, 5 mM CaCl2 and 1 mM 4 aminophenyl mercuric acetate (APMA) for 2-4 hours at 37°C. To dissolve APMA, make a 10 mM stock solution in 0.05 M NaOH. Approximately 90% of proenzyme MMP-3 is activated with a 4 hour incubation at 37°C using 1 mM APMA.
Storage Avoid freeze/thaw
≤ -70°C
Do Not Freeze Ok to freeze
Special InstructionsFollowing reconstitution, aliquot into siliconized vials and freeze (-70°C).
Toxicity Standard Handling
ReferencesSole, S., et al. 2004. J. Neuropathol. Exp. Neurol. 63, 338.
Galazka, G., et al. 1996. Biochem. 35, 11221.
Cottam, D.W. and Rees, R.C. 1993. Intl. J. Oncol. 2, 861.
Stetler-Stevenson, W.G., et al. 1993. FASEB J. 7, 1434.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Woessner, J.F. 1991. FASEB J. 5, 2145.
Liotta, L.A. and Stetler-Stevenson, W.G. 1990. In Seminars in Cancer Biology, ed. M.M. Gottesman. Vol. 1, 99.

Zymography References
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 References
Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259.
Netzel-Arnett, S., et al. 1991. Anal. Biochem. 195, 86.
Application referencesZYMOGRAPHY REFERENCES 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 REFERENCE Xia, T., et al. 1996. Biochim. Biophys. Acta. 1293, 259. Netzel-Arnett S, et al. 1991. Anal. Biochem. 195, 86.