Recombinant Human Serpin B3/SSCA1 Protein, CF Summary
Asn2-Pro390, with an N-terminal Met and 6-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||Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.|
|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 MES, 5 mM DTT, pH 6.0
- Recombinant Human Serpin B3/SCCA1 (rhSerpin B3) (Catalog # 6528-PI)
- Recombinant Human Cathepsin L (rhCathepsin L) (Catalog # 952-CY)
- Substrate: Z-Leu-Arg-AMC (Catalog # ES008)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Dilute rhCathepsin L to 40 µg/mL in Assay Buffer.
- Incubate 40 µg/mL rhCathepsin L for 15 minutes on ice to activate.
- Dilute activated rhCathepsin L to 0.2 µg/mL in Assay Buffer.
- Prepare a curve of rhSerpin B3 (MW = 45386 Da) in Assay Buffer. Make the following serial dilutions: 1000, 200, 50, 35, 25, 15, 5, 2 and 1 nM.
- Combine 25 µL of each dilution with 25 µL of the 0.2 µg/mL rhCathepsin L. Include an enzyme control containing Assay Buffer in place of rhSerpin B3.
- Incubate reaction mixtures at 37 °C for 15 minutes.
- Dilute reactions by adding 200 µL Assay Buffer to each.
- Dilute Substrate to 20 µM in Assay Buffer.
- Load into a black well plate 50 µL of the diluted incubated mixtures and start the reaction by adding 50 µL 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% inhibition concentration (IC50) value for rhSerpin B3 by plotting RFU/min vs. concentration with 4-PL fitting.
- Calculate the specific activity for rhCathepsin L at each point 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).
- rhCathepsin L: 0.001 µg
- rhSerpin B3: 50, 10, 2.5, 1.75, 1.25, 0.75, 0.25, 0.1, and 0.05 nM
- Substrate: 10 μM
Background: Serpin B3/SCCA1
Serpin B3, also known as squamous cell carcinoma antigen-1 (SCCA-1), is a member of the serpin superfamily of serine protease inhibitors (1). Serpins primarily inhibit serine proteases and some are known to inhibit caspases and papain-like cysteine proteases (1). Serpin B3 belongs to the subgroup ovalbumin-related serpins which are involved in the regulation of apoptosis, inflammation, angiogenesis and embryogenesis (2). Serpin B3 was initially isolated from human cervical squamous cell tissue. It is a tumor marker for squamous cell carcinomas in various areas including the cervix, head, neck and lung (3). Unlike typical serpins, Serpin B3 shows inhibitory activity against papain-like lysosomal cysteine proteases Cathepsin K, L, and S, while lacking inhibitory activity against serine proteases (4).
- Law, R. et al. (2006) Genome Biol. 7:216.
- Benarafa, C. and Remold-O’Donnell, E. (2005) Proc. Natl. Acad. Sci. 102:11367.
- Higgens, W.J. et al. (2010) J. Biol. Chem. 285:3722.
- Schick, C. et al. (1998) Biochemistry. 37:5258.
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Fluorogenic Peptide Substrates
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