Recombinant Human Dopa Decarboxylase/DDC Protein, CF Summary
Met1-Glu480, with a C-terminal 6-His tag
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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 MES, NaCl 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 Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.2
- Recombinant Human Dopa Decarboxylase/DDC (rhDopa) (Catalog # 3564-DC)
- Pyridoxal 5’-Phosphate hydrate (Sigma, Catalog # P9255)
- 3,4-dihydroxy L-phenylalanine (L-DOPA) (Sigma, Catalog # D9628)
- 2,4,6-Trinitrobenzene sulfonic acid (TNBS), 5% picryl solution (Sigma, Catalog # P2297)
- Benzene (Sigma, Catalog # 319953)
- 96 well clear UV-transparent microplate (Corning, Catalog # 3635)
- Plate Reader (Model: Spectramax Plus by Molecular Devices) or equivalent
- Prepare 5 mM stock solutions of L-DOPA and pyridoxal phosphate in deionized water. Use vigorous vortexing and mixing to get both reagents into solution.
- Dilute rhDopa to 5 ng/µL in Assay Buffer.
- Combine 160 µL of 5 ng/μL rhDopa with 20 µL 5 mM L-DOPA, 4 µL 5 mM pyridoxal phosphate, and 16 µL Assay Buffer. Include a control containing 20 µL 5 mM L-DOPA, 4 µL 5 mM pyridoxal phosphate, and 176 µL Assay Buffer without any rhDopa.
- Incubate for 30 minutes at 37 °C.
- Stop the reaction by heating for 2 minutes at 95 - 100 °C. After heating, cool on ice.
- Add 10 µL of 5% TNBS to each tube and vortex.
- Incubate for 20 minutes at 37 °C. Ensure that all samples undergo derivitization for the same length of time, stagger the addition of TNBS and the benzene extraction (next step) if necessary.
- In a fume hood, add 300 µL of benzene to each reaction in the same order the 5% TNBS was added. Vortex for 30 seconds.
- Centrifuge all reactions at 13,000 rpm in a microcentrifuge for 5 minutes to separate the aqueous and organic solvents.
- Carefully pipet out 200 µL of the benzene phase (the upper layer) of each reaction and load into a UV-transparent microplate. It is recommended that the microplate be kept covered as much as possible to minimize evaporation of the benzene.
- Read at an absorbance of 340 nm 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 dopamine (3,4-dihydroxyphenylethylamine) (Sigma, Catalog # H8502).Per Reaction:
- rhDopa: 0.800 µg
- L-DOPA: 500 µM
- Pyridoxal Phosphate: 100 µM
Background: Dopa Decarboxylase/DDC
Dopa Decarboxylase (DDC), also known as aromatic amino acid decarboxylase, is a group II decarboxylase (1). The enzyme catalyzes the decarboxylation of aromatic L-amino acids to produce the corresponding amines. DDC produces the neurotransmitters dopamine and serotonin from L-Dopa and L-5-hydroxytryptophan, respectively. The inhibition of DDC could be used for the treatment of schizophrenia and Parkinson's disease (2).
- Sandmeier, E. et al. (1994) Eur. J. Biochem. 221:997.
- Bertoldi, M. et al. (1996) J. Biol. Chem. 271:23954.
Citation for Recombinant Human Dopa Decarboxylase/DDC 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.
1 Citation: Showing 1 - 1
Rapid, high-yield enzymatic synthesis of n.c.a. 6-[18F]fluorodopamine (6-[18F]FDA) for in vivo application
Authors: K Bamminger, J Raitanen, G Karanikas, S Rasul, L Nics, M Mitterhaus, W Wadsak, M Hacker, V Pichler, C Vraka
Sample Types: Recombinant Proteins
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