Recombinant Mouse Cystatin E/M Protein, CF Summary
Glu29-Val149 & Thr35-Val149, both with a C-terminal 10-His tag
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.
|Formulation||Lyophilized from a 0.2 μm filtered solution in MES and NaCl.|
|Reconstitution||Reconstitute at 100 μg/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 Buffer: 50 mM Tris, pH 7.0
- Dithiothreitol (DTT) (Sigma, Catalog # D0632), 1 M stock in deionized water
- Recombinant Mouse Cystatin E/M (rmCystatin E/M) (Catalog # 1284-PI)
- Papain (Sigma, Catalog # P-4762)
- Substrate: 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 the Assay Buffer on ice.
- Dilute Papain to 100 µg/mL in Assay Buffer with 5 mM DTT.
- Incubate at room temperature for 15 minutes.
- Dilute activated Papain to 2 ng/µL in Assay Buffer.
- Prepare a curve of rmCystatin E/M (MW: 18,954 Da) in Assay Buffer. Make the following serial dilutions: 2000, 1000, 500, 250, 125, 93.75, 62.5, 46.875, 31.25, 15.63, 7.8, and 3.9 nM.
- Combine equal volumes of the rmCystatin E/M curve dilutions and 2 ng/µL active Papain. Include a control (in duplicate) containing equal volumes of Assay Buffer and 2 ng/µL Papain without adding any rmCystatin E/M.
- Incubate mixtures at 37 °C for 15 minutes.
- Make a five-fold dilution of reaction mixture with Assay Buffer.
- Dilute Substrate to 200 µM in Assay Buffer.
- Load into a black well plate 50 µL of the incubated mixtures and start the reaction by adding 50 µL of 200 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) for rmCystatin E/M 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 (Sigma, Catalog # A-9891).
- Papain: 0.010 µg
- rmCystatin E/M curve: 100, 50, 25, 12.5, 6.25, 4.7, 3.1, 2.3, 1.57, 0.78, 0.39, and 0.195 nM
- Substrate: 100 µM
Background: Cystatin E/M
Cystatin E/M is a member of family 2 of the cystatin superfamily (1‑6). It is required for viability and for correct formation of cornified layers in the epidermis and hair follicles, as ichq mice with a null mutation in the cystatin E/M gene have defects in epidermal cornification and die between 5 and 12 days of age (7). Cystatin E/M expression and function may not be limited to cutaneous epithelia. For example, it is found in rat brain and is induced during neuronal cell differentiation (8). The mouse cystatin E/M has not been characterized and is shown here to inhibit papain. In addition to being a cysteine protease inhibitor, the human cystatin E/M is also a substrate for transglutaminases (9).
- Carninci, P. and Y. Hayashizaki (1999) Meth. Enzymol. 303:19.
- Carninci, P. et al. (2000) Genome Res. 10:1617.
- Shibata, K. et al. (2000) Genome Res. 10:1757.
- Kawai, J. et al. (2001) Nature 409:685.
- Sotiropoulou, G. et al. (1997) J. Biol. Chem. 272:903.
- Ni, J. et al. (1997) J. Biol. Chem. 272:10853.
- Zeeuwen, P.L. et al. (2002) Hum. Mol. Genet. 11:2867.
- Hong, J. et al. (2002) J. Neurochem. 81:922.
- Zeeuwen, P.L. et al. (2001) J. Invest. Dermatol. 116:693.
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Fluorogenic Peptide Substrates
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