Measured by its ability to inhibit papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009). The IC50 value is <3.4 nM, as measured under the described conditions.
Source
E. coli-derived human Cystatin A protein Ile2-Phe98, with an N-terminal Met and 6-His tag
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Note
<1.0 EU per 1 μg of the protein by the LAL method.
Applications/Dilutions
Dilutions
Inhibition Activity
Theoretical MW
12 kDa. Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
12 kDa, reducing conditions
Publications
Read Publications using 1407-PI in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -20 to -70 °C as supplied.
3 months, -20 to -70 °C under sterile conditions after reconstitution.
Buffer
Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Reconstitution Instructions
Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
Assay Procedure
Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
Assay Buffer: 50 mM Tris, pH 7.0
Recombinant Human Cystatin A (rhCystatin A) (Catalog # 1407-PI)
Papain (Sigma, Catalog # P4762)
Substrate: N-Carbobenzyloxy-Phe-Arg-7-amido-4-methylcoumarin (Z-Phe-Arg-AMC) (Catalog # ES009), 10 mM stock in DMSO
F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
Chill Activation Buffer on ice.
Dilute Papain to 100 µg/mL in Activation Buffer.
Incubate at room temperature for 15 minutes.
Prepare a dilution curve of rhCystatin A (MW: 11,822 Da) in Assay Buffer. Make the following serial dilutions: 400, 200, 100, 66.7, 44.4, 29.6, 19.8, and 6.58 nM.
Dilute activated papain to 2.33 µg/mL in Assay Buffer.
Mix equal volumes of the rhCystatin A curve dilutions and diluted activated Papain. Include a control (in duplicate) containing Assay Buffer and the diluted active Papain.
Incubate mixtures at room temperature for 15 minutes.
Dilute Substrate to 200 µM in Assay Buffer.
Perform a five-fold dilution with Assay Buffer to the incubated mixture of rhCystatin A and Papain.
Load 50 µL of diluted incubated mixture into a black well plate, and start the reaction by adding 50 µL of 200 μM Substrate.
Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
Derive the 50% inhibiting value (IC50) for rhCystatin A by plotting RFU/min (or specific activity) vs concentration with 4-PL fitting.
The specific activity for Papain 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 (AMC) (Sigma, Catalog # A-9891).
Per Well:
Papain: 0.01165 µg
Substrate: 100 µM
rhCystatin A curve: 20, 10, 5, 3.34, 2.22, 1.48, 0.99, and 0.329 nM
Notes
This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.
Alternate Names for Recombinant Human Cystatin A Protein, CF
AREI
CSTA
cystatin A (stefin A)
Cystatin A
cystatin AS
cystatin-A
Stefin A
STF1
STF1Cystatin-AS
STFA
STFAStefin-A
Background
Cystatin A, also called stefin A, Cystatin AS or keratolinin, is a member of family 1 of the cystatin superfamily (1, 2). Like Cystatin B, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L (3). For example, immunohistochemical analysis of Cystatin A and cathepsin L is a useful indicator for malignancy in human epidermal keratinocytes (4). The ratio of cathepsin B and Cystatin A can be used in the differential diagnosis and treatment of patients with prostate carcinoma (5). The human Cystatin A consists of 98 amino acids (1).
Kartasova, T. et al. (1987) Nucleic Acids Res. 15:5945.
Abrahamson, M. (1994) Methods Enzymol. 244:685.
Jenko, S. et al. (2003) J. Mol. Biol. 326:875.
Palungwachira, P. et al. (2002) J. Dermatol. 29:573.
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