Recombinant Human Biotinidase/BTD Protein, CF Summary
Product Specifications
Ala42-Asp543, with a C-terminal 6-His tag
Analysis
Product Datasheets
Carrier Free
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.
7839-BT
| Formulation | Supplied as a 0.2 μm filtered solution in NaH2PO4, NaCl, EDTA and DTT. |
| Shipping | The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below. |
| Stability & Storage: | Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
|
Assay Procedure
- Assay Buffer: 50 mM Tris, 0.2 M NaCl, 0.1% Triton, pH 7.5
- Recombinant Human Biotinidase/BTD (rhBTD) (Catalog # 7839-BT)
- (+)-Biotin 4-Nitrophenyl ester (BNP) (Sigma, Catalog # 861650), 50 mM stock in DMSO
- Dimethyl Sulfoxide (DMSO) (Sigma, Catalog # 34869)
- 96-well Clear Plate (Catalog # DY990)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhBTD to 20 µg/mL in Assay Buffer.
- Dilute room temperature BNP to 1 mM in DMSO. Mix well.
- Load 50 µL of 20 µg/mL rhBTD in a clear strip well plate, and start the reaction by adding 50 µL of 1 mM BNP. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 1 mM BNP.
- Incubate sealed plate at room temperature for 10 minutes in the dark.
- Read at 405 nm (absorbance) in endpoint mode.
- Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard p-Nitrophenol (Sigma, Catalog # 241326). Per Well:- rhBTD: 1 µg
- BNP: 0.5 mM
Reconstitution Calculator
Background: Biotinidase/BTD
Biotinidase (BTD) is a member of the nitrilase superfamily, which consists of 12 families of nitrilases, amidases, carbamylases, and N‑acyltrasferases (1). It is a thiol hydrolase releasing biotin from biotinamide, biotin‑lysine, biotin‑peptide conjugates and biotin methylester. It is expressed in most mammalian tissues, with high activity being present in liver, kidney, serum, intestine, and adrenal glands (2). BTD with two other proteins, sodium-dependent multivitamin transporter, and holocarboxylase synthetase, play major roles in the homeostasis of biotin (3). BTD contributes to the homeostasis through the intestinal release of free biotin from digested biotin‑containing proteins and plasma transport and the recycling of biotin from breakdown products of biotinylated carboxylases. BTD deficiency can lead to a decrease in biotin bioavailability due to failure in releasing biotin from dietary proteins. It can be caused by gene mutations or from decreased secretion of BTD into the intestinal lumen (4). A recent study indicates that the BTD level in human plasma is a potential biomarker for the detection of breast cancer (5).
- Pace, H. C. and C. Brenner (2001) Genome. Biol. 2:reviews/0001.1.
- Chauhan, J. and K. Dakshinamurt (1986) J. Biol. Chem. 261:4286.
- Wolf, B. (2005) J Nur Biochem. 16:441.
- Zempleni, J. et al. (2008) Expert Rev Endocrinol Metab. 3:715.
- Kang, U. B. et al. (2010) BMC Cancer 10:114.
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