Recombinant Mouse Cystatin B Protein, CF

Catalog #: 1409-PI
1 Citation Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
1409-PI-050

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Summary Product Datasheets Carrier Free Data Images Reconstitution Calculator Background Related Research Areas

Recombinant Mouse Cystatin B Protein, CF Summary

Product Specifications

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 papain cleavage of a fluorogenic peptide substrate Z-FR-AMC (Catalog # ES009). The IC₅₀ value is <15 nM, as measured under the described conditions.

Source

E. coli-derived mouse Cystatin B protein
Met1-Phe98, with an N-terminal Met and 6-His tag

Accession #

Q62426

N-terminal Sequence
Analysis

Met

Structure / Form

Monomer and a disulfide-linked multimer

Predicted Molecular Mass

12 kDa (monomer)

SDS-PAGE

13 kDa, reducing conditions

Product Datasheets

1409-PI

Product Datasheet

COA

SDS

Carrier Free

What does CF mean?

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.

What formulation is right for me?

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.

1409-PI

Formulation	Lyophilized from a 0.2 μm filtered solution in Tris and NaCl.
Reconstitution	Reconstitute at 100 μg/mL in sterile 25 mM Tris and 100 mM NaCl, pH 7.5.
Shipping	The product is shipped at ambient temperature. Upon receipt, store it immediately at the temperature recommended below.
Stability & Storage:	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.

Assay Procedure

Materials

Activation Buffer: 50 mM Tris, 5 mM DTT, pH 7.0
Assay Buffer: 50 mM Tris, pH 7.0
Recombinant Mouse Cystatin B (rmCystatin B) (Catalog # 1409-PI)
Papain (Sigma, Catalog # P4762)
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

Dilute Papain to 100 µg/mL in Activation Buffer.
Incubate at room temperature for 15 minutes.
Prepare a curve of rmCystatin B (MW: 11,991 Da) in Assay Buffer. Make the following serial dilutions: 800, 400, 200, 150, 100, 50, 25 and 12.5 nM.
Dilute activated Papain to 2.4 µg/mL in Assay Buffer.
Combine 20 µL of 2.4 µg/mL Papain with 20 µL of rmCystatin B curve dilutions. Include two controls containing 20 µL Assay Buffer with 20 µL of 2.4 µg/mL Papain.
Incubate at room temperature for 10 minutes.
After incubation, dilute the rmCystatin B curve 5-fold by adding 160 µL of Assay Buffer.
Dilute Substrate to 200 µM in Assay Buffer.
In a plate, load 50 µL of the diluted rmCystatin B curve, and the start the reaction by adding 50 µL of 200 µM Substrate to wells.
Read at excitation and emission wavelengths of 380 nm and 460 nm, respectively, for 5 minutes in kinetic mode.
Derive the 50% inhibition concentration (IC₅₀) value for rmCystatin B 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 V_max* (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: 0.012 µg
Substrate: 100 µM
rmCystatin B curve: 40, 20, 10, 7.5, 5, 2.5, 1.25 and 0.625 nM

Reconstitution Calculator

Background: Cystatin B

Cystatin B, also called stefin B or liver thiol proteinase inhibitor, is a member of family 1 of the cystatin superfamily (1). Like Cystatin A, it is an intracellular inhibitor regulating the activities of cysteine proteases of the papain family such as cathepsins B, H and L (2). Cystatin B-deficient mice have increased expression of proteolysis, apoptosis and glial activation genes, which is consistent with the pathology found in the mouse model of human progressive myoclonus epilepsy (EPM1) (3). The mouse Cystatin B consists of 98 amino acid residues (4).

References

Abrahamson, M. (1994) Methods Enzymol. 244:685.
Pol, E. and I. Bjork (1999) Biochemistry 38:10519.
Lieuallen, K. et al. (2001) Hum. Mol. Genet. 10:1867.
Pennacchio, L.A. and R.M. Myers (1996) Genome Res. 6:1103.

Entrez Gene IDs

1476 (Human); 13014 (Mouse)

Alternate Names

CPI-B; CST6cystatin B (liver thiol proteinase inhibitor)10STFBcystatin-B; CSTB; cystatin B (stefin B); Cystatin B; EPM1; Liver thiol proteinase inhibitor; PME; Stefin B; stefin-B

Related Research Areas

Cysteine Proteases and Regulators

Citation for Recombinant Mouse Cystatin B 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.

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