Recombinant Human COMT Protein, CF Summary
Product Specifications
Gly52-Pro271, with an N-terminal Met and 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.
7386-MT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and TCEP. |
| 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: 20 mM Tris, 2 mM MgCl2, pH 8.0
- Recombinant Human COMT (rhCOMT) (Catalog # 7386-MT)
- Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water
- Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
- Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
- S-adenosylmethionine (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO in deionized water
- Catechol (Sigma, Catalog # C9510), 0.2M stock in deionized water
- ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates Inc., Catalog # T-003), 10 mM stock in DMSO
- DMSO (Sigma, Catalog # 154938)
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute the stock of reduced glutathione to 40 µM (40 pmol/µL) in Assay Buffer. This is the first point of the standard curve.
- Continue standard curve by performing six ½ serial dilutions of the 40 µM glutathione in Assay Buffer. The standard curve has a range of 31.25 to 2000 pmol per well.
- Dilute rhCOMT to 12 µg/mL in Assay Buffer.
- Dilute rhAHCY to 240 µg/mL in Assay Buffer.
- Dilute rhADA to 200 µg/mL in Assay Buffer.
- Prepare Enzyme Mixture by combining equal volumes of diluted rhCOMT, rhAHCY and rhADA. For a Control, use Assay Buffer in place of rhCOMT.
- Dilute S-adenosylmethionine to 800 µM in Assay Buffer.
- Dilute catechol to 4 mM in Assay Buffer.
- Prepare Substrate Mixture by combining equal volumes of diluted S-adenosylmethionine and catechol.
- Load into plate 50 µL of each dilution of the standard curve. Include a curve blank containing 50 µL of Assay Buffer.
- Load into plate 25 µL of the Control and Enzyme Mixture (step 6).
- Start the reaction by adding 25 µL of Substrate Mixture (step 9) to the wells, excluding standard curve and curve blank.
- Cover microplate and incubate at 37 °C for 30 minutes.
- Dilute ThioGlo® to 100 µM in DMSO.
- After incubation, add 50 µL of 100 µM ThioGlo® to each well.
- Incubate at room temperature for 5 minutes in the dark.
- Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Thiol produced* (pmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the reduced glutathione standard curve using linear fitting and adjusted for Substrate Blank.
Per Well:- rhCOMT: 0.100 µg
- rhAHCY: 2 µg
- rhADA: 1.667 µg
- S-adenosylmethionine: 100 µM
- Catechol: 0.5 mM
- ThioGlo®: 50 µM
Reconstitution Calculator
Background: COMT
Catechol-O-methyltransferase (COMT) catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to either the meta- or the para-hydroxyl group of the catechol ring from a variety of endogenous and exogenous catechol substrates including the neurotransmitters dopamine, epinephrine, and norepinephrine (1). Thus, it plays important roles in one of the major degradative pathways of the catecholamine transmitters, and in the metabolism of catechol drugs used in the treatment of hypertension, asthma, and Parkinson’s disease. In tissues, COMT is produced as two distinct forms, a soluble protein (residues 52-271) and a membrane-bound protein that possesses the N-terminal transmembrane domain (2). A common single-nucleotide polymorphism results in substitution of methionine for valine at position 158 of membrane-bound COMT and position 108 at soluble COMT (3). This amino acid change results in a more unstable and three- to four-fold less active 108/158Met form (4). Since COMT activity is the major regulator of dopamine function in the prefrontal brain region, the Val108/158Met variation is considered to be associated with increased risk of a wide spectrum of mental disorders (5).
- Zhu, B. T. (2002) Curr. Drug Metab. 3:321.
- Lotta, T. et al. (1995) Biochemistry 34:4202.
- Lachman. H. M. et al. (1996) Pharmacogenetics 6:243.
- Rutherford, K. et al. (2008) Biochim. Biophys. Acta 1784:1098.
Citation for Recombinant Human COMT 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
-
N-methylation of BI 187004 by thiol S-methyltransferase
Authors: HH Maw, X Zeng, S Campbell, ME Taub, AM Teitelbaum
Drug Metab. Dispos., 2018-03-07;0(0):.
Species: Human
Sample Types: Recombinant Protein
Applications: Bioassay
FAQs
No product specific FAQs exist for this product, however you may
View all Proteins and Enzyme FAQsReviews for Recombinant Human COMT Protein, CF
There are currently no reviews for this product. Be the first to review Recombinant Human COMT Protein, CF and earn rewards!
Have you used Recombinant Human COMT Protein, CF?
Submit a review and receive an Amazon gift card.
$25/€18/£15/$25CAN/¥75 Yuan/¥2500 Yen for a review with an image
$10/€7/£6/$10 CAD/¥70 Yuan/¥1110 Yen for a review without an image
