Recombinant Human Cystatin S Protein, CF Summary
Product Specifications
Ser21-Ala141, 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.
1296-PI
| Formulation | Lyophilized from a 0.2 μm filtered solution in MES and NaCl. |
| Reconstitution | Reconstitute at 1 mg/mL in sterile 25 mM MES, 150 mM NaCl, pH 6.5. |
| 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: 50 mM MES, 5 mM DTT, pH 6.0
- Assay Buffer: 50 mM MES, pH 6.0
- Recombinant Human Cystatin S (rhCystatin S) (Catalog # 1296-PI)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- Substrate: Z-Leu-Arg-AMC (Catalog # ES008), 2 mM stock in DMSO.
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhCathepsin L to 1 µg/mL in Activation Buffer.
- Incubate on ice for 15 minutes.
- After incubation, dilute activated rhCathepsin L to 0.5 µg/mL in Assay Buffer.
- Prepare a curve of rhCystatin S (MW: 15,550 Da) in Assay Buffer. Make the following serial dilutions: ≥45.7 µM (stock concentration should be ≥ 0.711 mg/mL), 30.5, 20.3, 13.5, 9, 6, 4, and 2.68 µM.
- Combine equal volumes of the rhCystatin S curve dilutions and the diluted active rhCathepsin L. Include a control (in duplicate) containing Assay Buffer and the diluted active rhCathepsin L.
- Incubate mixtures at 37 °C for 15 minutes.
- Perform a 1/5 dilution of each reaction mixture with Assay Buffer.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load 50 µL of the incubated mixtures in a plate, and start the reaction by adding 50 µL of 20 µM Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes. - Derive the 50% inhibiting concentration (IC50) of rhCystatin S by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.
- The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):
|
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).
Per Well:- rhCathepsin L (MW: 25524 Da): 0.0025 µg
- rhCystatin S curve: ≥2.286 µM (variable based on stock concentration), 1.525, 1.015, 0.675, 0.45, 0.3, 0.2, and 0.134 µM.
- Substrate: 10 µM
Reconstitution Calculator
Background: Cystatin S
Cystatin S is a member of family 2 of the cystatin superfamily (1). Like cystatins SA and SN, it is produced by the salivary gland and secreted largely in the submandibular/sublingual saliva (2). Unlike cystatins SA and SN, Cystatin S is not a potent inhibitor of cysteine proteases such as papain and some cathepsins (3). Cystatin S is partially phosphorylated at Ser21 and Ser23 in saliva (4). The functions of Cystatin S are largely unknown. However, it is able to bind more calcium and bind more rapidly to carbonated apatite than cystatins SA or SN, indicating that Cystatin S may be involved in the mineral balance of the tooth (3).
- Abrahamson, M. (1994) Methods Enzymol. 244:685.
- Baron, A.C. et al. (1999) Oral. Dis. 5:344.
- Baron, A. et al. (1999) Oral Dis. 5:234.
- Isemura, S. et al. (1991) J. Biochem. 110:648.
Citations for Recombinant Human Cystatin S Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
2
Citations: Showing 1 - 2
Filter your results:
Filter by:
-
Cystatin M/E is a high affinity inhibitor of cathepsin V and cathepsin L by a reactive site that is distinct from the legumain-binding site. A novel clue for the role of cystatin M/E in epidermal cornification.
Authors: Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K, Watts C, Reinheckel T, Schalkwijk J, Zeeuwen PL
J. Biol. Chem., 2006-03-24;281(23):15893-9.
Species: Human
Sample Types: Recombinant Protein
Applications: Enzyme Assay -
Characterization of cathepsin L secreted by Sf21 insect cells.
Authors: Johnson GD, Jiang W
Arch. Biochem. Biophys., 2005-10-21;444(1):7-14.
Applications: Enzyme Assay
FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human Cystatin S Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human Cystatin S Protein, CF and earn rewards!
Have you used Recombinant Human Cystatin S Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
