Recombinant Rat Cathepsin C/DPPI Protein, CF Summary
Product Specifications
Asp25-Leu462, 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.
8285-CY
| Formulation | Supplied as a 0.2 μm filtered solution in MES 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
- Activation Buffer: 25 mM MES, 5 mM DTT, pH 5.5
- Assay Buffer: 25 mM MES, 50 mM NaCl, 5 mM DTT, pH 6.0
- Recombinant Rat Cathepsin C/DPPI (rrCathepsin C) (Catalog # 8285-CY)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- Substrate: Gly-Arg-AMC (Bachem, Catalog # I-1215)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rrCathepsin C to 20 µg/mL in Activation Buffer.
- Dilute rhCathepsin L to 2 µg/mL in Activation Buffer.
- Combine equal volumes of diluted rrCathepsin C and diluted rhCathepsin L for final concentrations of 10 µg/mL and 1 µg/mL respectively.
- Incubate at room temperature for 30 minutes to activate rrCathepsin C.
- Dilute activated rrCathepsin C to 0.02 µg/mL in Assay Buffer.
- Dilute Substrate to 100 µM in Assay Buffer.
- Load 50 µL of the 0.02 µg/mL rrCathepsin C into a plate, and start the reaction by adding 50 µL of 100 µM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 100 µM Substrate.
- Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, 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 Substrate Blank
**Derived using calibration standard 7-amino, 4-Methyl Coumarin (Sigma, Catalog # A-9891).
- rrCathepsin C: 0.001 µg
- Substrate: 50 µM
Reconstitution Calculator
Background: Cathepsin C/DPPI
Cathepsin C (CTSC), also known as
dipeptidyl-peptidase I (DPPI), is a chloride-dependent cysteine protease in the
papain family (1-3). It sequentially removes dipeptides from the free N-terminus
of proteins and peptides. It has broad specificity except that it does not
cleave proteins with a basic amino acid (Arg or Lys) in the N-terminal position
or with a proline on either side of the scissile bond. Cathepsin C is
synthesized with a large propeptide followed by a mature region, which is
further cleaved into heavy and light chains during processing. The N-terminal
region of the propeptide is known as the exclusion domain. It is present in the
mature, active enzyme and regulates access of substrates to the active site (4).
Cathepsin C is widely expressed and plays a major role in lysosomal
degradation and enzyme activation. It activates granule serine proteases in
cytotoxic T lymphocytes and natural killer cells (Granzymes A and B),
mast cells (Tryptase and Chymase), and neutrophils (Cathepsin G and Elastase)
by removing their N-terminal activation dipeptides (5).
- Turk, B.E. et al. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp. 1192, Elsevier Academic Press, San Diego.
- Minarowska, A. et al. (2012) Folia Histochem. Cytobiol. 50:20.
- Ishidoh, K. et al. (1991) J. Biol. Chem. 266:16312.
- Turk, D. et al. (2001) EMBO J. 20:6570.
- Dahl, S.W. et al. (2001) Biochemistry 40:1671.
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