Product Details
| Alternative Name: | Procathepsin O, Procathepsin O2, Procathepsin X |
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| MW: | ~35kDa |
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| Source: | Produced in E. coli (aa 19-329). Methionine was introduced at aa 18 to create a new N-terminal sequence (MEEIL). |
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| EC: | 3.4.22.38 |
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| UniProt ID: | P43235 |
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| Concentration: | 150μg/ml |
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| Formulation: | Liquid. In 25mM TRIS, pH 8.0, containing 500mM sodium chloride. |
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| Purity: | ≥95% (SDS-PAGE) |
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| Specific Activity: | ≥1'000mU/mg protein. One unit is defined as the amount of enzyme that hydrolyzes 1µmol of substrate per min. at 37°C, pH 5.5. |
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| Shipping: | Dry Ice |
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| Long Term Storage: | -80°C |
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| Use/Stability: | Stable for several months when stored at -80°C. After activation, cathepsin is highly autoproteolytic. When not used immediately, add MMTS (Methyl methanethiosulfonate) to 1mM final concentration for stabilization and shock freeze in liquid nitrogen. Do not store dilutions of the enzyme. |
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| Handling: | Avoid freeze/thaw cycles. |
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| Scientific Background: | Member of the papain cysteine proteinase family identified as the predominant proteinase responsible for the resorption of the bone matrix. |
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| Technical Info/Product Notes: | Inhibitors: Cathepsin K is inhibited by leupeptin (Prod. No. ALX-260-009) (IC50=70nM), E-64 (IC50=5nM) or cystatine. Minimal effects exhibit pepstatin, phenylmethylsulfonyl fluoride (Prod. No. ALX-270-184), inhibitors of aspartyl and serine proteases. No inhibition was observed by addition of EDTA or phenanthroline, classical inhibitors of metalloproteases. |
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| Protocol: |
Activity: Activity of mature protein was measured in 50mM MES, pH 5.5, containing 2.5mM EDTA and 5mM DTT. Assay was initiated by addition of substrate Z-Phe-Arg-AMC (Prod. No. BML-P139) to a final concentration of 100µM (stock solution 10mM in DMSO). Activity was monitored by the increase of fluorescence accompanying release of the product 7-amino-4-methylcoumarin (AMC) (excitation wavelength of 360nm; emission wavelength of 460nm).
Activation: Activate by adjusting procathepsin K to pH 4.0 by adding an equal volume of 100mM sodium acetate, pH 3.9, containing 10mM DTT, 5mM EDTA and incubate for 40 min. at room temperature. MMTS (methyl methanethiosulfonate) modifies cysteines by attaching its relatively small, uncharged thiomethyl blocking group to reactive sulfhydryl groups. This reversible reaction arrests the autoproteolytical process. Activity can be restored to nearly unmodified level by adding L-cysteine (~3M excess over MMTS). |
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| Regulatory Status: | RUO - Research Use Only |
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Product Literature References
Autocatalytic activation of human cathepsin K: M.S. McQueney, et al.; J. Biol. Chem.
272, 13955 (1997),
Abstract;
Full Text
Reversible modification of the sulfhydryl groups of Escherichia coli succinic thiokinase with methanethiolating reagents, 5.5’-Dithio-bis(2-nitrobenzoic acid), p-hydroxymercuribenzoate, and ethylmercurithiosalicylate: J.S. Nishimura, et al.; Arch. Biochem. Biophys.
170, 461 (1975),
Abstract;
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