Recombinant Mouse Cathepsin A/Lysosom Carboxypeptidase A, CF
Recombinant Mouse Cathepsin A/Lysosom Carboxypeptidase A, CF Summary
Product Specifications
Ala24-Tyr474, with a C-terminal 10-His tag
Analysis
Product Datasheets
Carrier Free
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
9789-SE
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| Shipping | The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 25 mM MES, 0.5 mM TCEP, pH 5.5
- Recombinant Mouse Cathepsin A/Lysosomal Carboxypeptidase A (rmCathepsin A) (Catalog # 9789-SE)
- Activator: Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- E 64 (Tocris, Catalog # 5208), 50 mM stock in DMSO
- Fluorogenic Peptide Substrate V: MCA-RPPGFSAFK-(DNP)-OH (Catalog # ES005), 2 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmCathepsin A to 100 μg/mL in Assay Buffer.
- Dilute rhCathepsin L to 10 μg/mL in Assay Buffer.
- Combine equal volumes of rmCathepsin A and rhCathepsin L for final concentrations of 50 μg/mL and 5 μg/mL, respectively. Include a control containing Assay Buffer in place of rmCathepsin A (Cathepsin L Control).
- Incubate at room temperature for 30 minutes.
- Dilute E 64 to 20 μM in Assay Buffer.
- Combine equal volumes of activated rmCathepsin A and 20 μM E 64 to stop the reaction. Combine equal volumes of Cathepsin L Control and 20 μM E 64.
- Dilute activated rmCathepsin A to 3 μg/mL in Assay Buffer. Perform equivalent dilution on Cathepsin L Control.
- Dilute Substrate to 40 μM in Assay Buffer.
- Load into a plate 50 μL of 3 μg/mL rmCathepsin A and Cathepsin L Control, and start the reaction by adding 50 μL of 40 μM Substrate.
- Read plate at excitation and emission wavelengths of 320 nm and 405 nm, respectively, (top read) in kinetic mode for 5 minutes.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU) |
| amount of enzyme (µg) |
*Adjusted for Cathepsin L Control.
**Derived using calibration standard Mca-PL-OH (Bachem, Catalog # M-1975).
- rmCathepsin A: 0.15 μg
- Substrate: 20 μM
Reconstitution Calculator
Background: Cathepsin A/Lysosomal Carboxypeptidase A
Cathepsin A/lyososomal carboxypeptidase A is a member of the serine carboxypeptidase family (1). Cathepsin A, also known as protective protein, is synthesized as a single-chain precursor and processed into heavy (32 kDa) and light (20 kDa) chains, which are linked by disulfide bonds. Cathepsin A is a multifunctional enzyme that expresses deaminidase and esterase activities at neutral pH and carboxypeptidase activity at acidic pH (2, 3). Cathepsin A is capable of hydrolyzing a variety of bioactive peptide hormones including endothelin and bradykinin making it a promising target in heart failure (2-5). A hereditary variant of cathepsin A results in cathepsin A-related arteriopathy with strokes and leukoencephalapathy (CARASAL) (6). In addition, Cathepsin A's association with beta -galactosidase ( beta -gal) and neuraminidase in a complex is essential for beta -gal stability and neuraminidase activation in the lysosomes. Inherited deficiency of Cathepsin A causes Galactosialidosis, a lysosomal storage disorder, characterized by a combined secondary deficiency of beta -gal and neuraminidase (7, 8).
- Pshezhetsky, A.V. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) p. 1923, Academic Press, San Diego.
- Hiraiwa, M. (1999) Cell. Mol. Life. Sci. 56:894.
- Schreuder, H.A. et al. (2014) Biochem. Biophys. Res. Commun. 445:451.
- Ruf, S. et al. (2013) Future Med. Chem. 5:399.
- Timur, Z.K. et al. (2016) Front. Mol. Biosci. 3:68.
- Bugiani, M. et al. (2016) Neurology 84:1777.
- Caciotti, A. et al. (2013) Ophanet. J. Rare Dis. 8:114.
- Annunziata, I. et al. (2017) Expert Opin. Orphan Drugs 5:131.
FAQs
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Where does Cathepsin A cleave Mca-RPPGFSAFK(Dnp)-OH Fluorogenic Peptide Substrate, Catalog # ES005?
Although the QC assay conditions provided on our recombinant Cathepsin A datasheets should favor carboxypeptidase activity, it is possible that there is more than one site in the ES005 peptide recognized and cleaved by Cathepsin A. We have not performed verification experiments to confirm the cleavage site preferred in our reaction conditions.
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