• Purity

  >95%, by SDS-PAGE under reducing conditions and visualized by silver stain

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to inhibit active Cathepsin L cleavage of a fluorogenic peptide substrate Z-LR-AMC (Catalog # ). The IC    ₅₀ value is <1.0 μM as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com  

• Source

  Mouse myeloma cell line, NS0-derived Ser21-Ala141, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ser21

• Predicted Molecular Mass

  15.6 kDa

• SDS-PAGE

  16 kDa, reducing conditions

Assay Procedure

Materials

• 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 # )

• Substrate: Z-Leu-Arg-AMC (Catalog # ), 2 mM stock in DMSO.

• F16 Black Maxisorp Plate (Nunc, Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute rhCathepsin L to 1 μg/mL in Activation Buffer.

2. Incubate on ice for 15 minutes.

3. After incubation, dilute activated rhCathepsin L to 0.5 μg/mL in Assay Buffer.

4. 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.

5. 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.

6. Incubate mixtures at 37 °C for 15 minutes.

7. Perform a 1/5 dilution of each reaction mixture with Assay Buffer.

8. Dilute Substrate to 20 μM in Assay Buffer.

9. 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.

10. Derive the 50% inhibiting concentration (IC₅₀) of rhCystatin S by plotting RFU/min (or specific activity) vs. concentration with 4-PL fitting.

11. The specific activity for rhCathepsin L at each point may be determined using the following formula (if needed):

     *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

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).

• References:

1. Abrahamson, M. (1994) Methods Enzymol. 244:685.

2. Baron, A.C. et al. (1999) Oral. Dis. 5:344.

3. Baron, A. et al. (1999) Oral Dis. 5:234.

4. Isemura, S. et al. (1991) J. Biochem. 110:648.

• Entrez Gene IDs:

  1472 (Human)

• Alternate Names:

  CST4; cystatin 4; Cystatin S; Cystatin-4; cystatin-S; cystatin-SA-III; MGC71923; Salivary acidic protein 1