Recombinant Human Dopamine beta-Hydroxylase Protein, CF
Recombinant Human Dopamine beta-Hydroxylase Protein, CF Summary
Product Specifications
Met1-Gly617, 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.
7376-AO
| Formulation | Supplied as a 0.2 μm filtered solution in Tris and NaCl. |
| 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 Procedure
- Assay Buffer: 0.1 M NaOAc, 0.5 µM CuCl2, pH 5.0
- Recombinant Human Dopamine beta ‑Hydroxylase (rhDBH) (Catalog # 7376-AO)
- Substrate Component 1: Tyramine (Sigma, Catalog # T2879), 100 mM stock in deionized water
- Substrate Component 2: N,N-Dimethyl-p-phenylenediamine (DMPD) (Sigma, Catalog # D4139), 500 mM stock in deionized water
- 96-well Clear Plate (Costar, Catalog # 92592)
- Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
- Dilute rhDBH to 1 ng/µL in Assay Buffer.
- Dilute both substrate components to 40 mM respectively in Assay Buffer.
- Combine equivalent volumes of substrate components for substrate mixture.
- Load 50 µL of the diluted rhDBH into a clear plate, and start the reaction by adding 50 µL of the Substrate mixture to wells. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL Substrate Mixture without any rhDBH.
- Read in kinetic mode for 5 minutes at an absorbance of 515 nm.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Vmax* (OD/min) x well volume (L) x 1012 pmol/mol |
| ext. coeff** (M-1cm-1) x path corr.*** (cm) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Using the extinction coefficient 5200 M-1cm-1
***Using the path correction 0.32 cm
Note: the output of many spectrophotometers is in mOD Per Well:
- rhDBH: 0.050 μg
- Tyramine: 10 mM
- DMPD: 10 mM
Reconstitution Calculator
Background: Dopamine beta-Hydroxylase
Dopamine beta -Hydroxylase (DBH), also known as dopamine beta -monooxygenase, belongs to the copper type II, ascorbate-dependent monooxygenase family. DBH is found within the neurosecretory vesicles of adrenal medullae and the large dense‑cored synaptic vesicles of the sympathetic nervous system as both membrane‑associated and soluble forms (1, 2). It catalyzes the conversion of dopamine to noradrenaline in sympathetic neurons, making it an important enzyme for catecholamine biosynthesis (3, 4). Mutations in the DBH gene that result in low DBH activity are a cause of noradrenaline deficiency (5). This recombinant human DBH is the secreted, soluble form.
- Dhawan, S. et al. (1987) J. Biol. Chem. 262:1869.
- Saxena, A. and P.J. Fleming (1983) J. Biol. Chem. 258:4147.
- Levin, E.Y. et al. (1960) J. Biol. Chem. 235:2080.
- Friedman, S. and S. Kaufman (1965) J. Biol. Chem. 240:4763.
- Kim, C.H. et al. (2002) Am. J. Med. Genet. 108:140.
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