Recombinant Human PNMT Protein, CF Summary
Product Specifications
Met1-Leu282, 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.
7854-MT
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, EDTA, Brij-35 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 Procedure
- Assay buffer: 25 mM MES, pH 6.0
- Hydrolysis buffer: 100 mM HEPES, pH 7.0
- Recombinant Human PNMT (rhPNMT) (Catalog # 7854-MT)
- Substrate: DL-Norepinephrine hydrochloride (Sigma, Catalog # A7256), 0.5 M stock in deionized water
- Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
- Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
- S-adenosylmethionine (AdoMet) (Sigma, Catalog # A7007), 20 mM stock in deionized water
- Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water.
- ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 mM stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhPNMT to 80 µg/mL in Assay Buffer.
- Create a Substrate Mixture containing 400 µM AdoMet and 1 mM DL-Norepinephrine hydrochloride in Assay Buffer.
- Combine equal volumes of rhPNMT and Substrate Mixture. As a Control, replace rhPNMT with Assay Buffer.
- Incubate reactions and control at room temperature for 30 minutes.
- Boil reactions and control at 95‑100 °C for 5 minutes. Cool samples on ice for 3 minutes.
- Create a Hydrolysis Mixture containing 25 µg/mL rhAHCY and 2.5 µg/mL rhADA in Hydrolysis Buffer.
- Add an equal volume of Hydrolysis Mixture to reactions and control.
- Incubate reactions and control at 37 °C for 1 hour.
- Prepare standard curve during incubation (step 7).
- Dilute Glutathione to 20 µM in Assay Buffer.
- Continue standard curve by performing six additional ½ dilutions in Assay Buffer.
- Load 25 µL of each curve point into the plate in triplicate. Include a standard blank of Assay Buffer only in triplicate.
- Add 25 µL of Hydrolysis Buffer to the curve wells.
- Load 50 µL of reaction(s) and control into empty wells in triplicate.
- Dilute ThioGlo to 100 µM in DMSO.
- Add 50 µL of 100 µM ThioGlo to all wells, including standard curve.
- Incubate sealed plate at room temperature for 5 minutes in the dark.
- Read at excitation and emission wavelengths of 380 nm and 445 nm (top read), respectively, in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Thiol produced* (pmol) |
| Incubation time (min) x amount of enzyme (µg) |
*Derived from the reduced glutathione standard curve using linear fitting and adjusted for Control.
Per Well:- rhPNMT: 1 µg
- DL-Norepinephrine hydrochloride: 125 µM
- AdoMet: 50 µM
- rhAHCY: 6.25 µg/mL
- rhADA: 0.625 µg/mL
- ThioGlo: 50 µM
- Glutathione Curve: 500, 250, 125, 62.4, 31.2, 15.6, 7.8 and 0 pmol
Reconstitution Calculator
Background: PNMT
Phenylethanolamine N-methyltransferase (PNMT) is the enzyme that catalyzes biosynthesis of epinephrine from norepinephrine using S-Adenosyl-L-Methionine (SAM) as the methyl donor (1). The enzyme is expressed in high levels in the adrenal medulla, where epinephrine is synthesized (2). It is also responsible for synthesis of epinephrine in the central nervous system as peripheral epinephrine does not cross the blood-brain barrier (3, 4). The function of PNMT has been implicated in regulation of blood pressure, respiration, body temperature, and in neurodegeneration observed in Parkinson’s disease and Alzheimer’s disease (5-7).
- Kirshner, N. et al. (1957) Biochim. Biophys. Acta. 24:658.
- Axelrod, J. (1962) J. Biol. Chem. 237:1657.
- Weil-Malherbe, H. et al. (1959) Science 129:1226.
- Ciaranello, R. D. et al. (1969) Nature 221:368.
- Saavedra, J. M. et al. (1976) Science 191:483.
- Rothballer, A. B. (1959) Pharmacol. Rev. 11:494.
- Mann, M. B. (2001) Am. J. Med. Genet. 105:312.
FAQs
-
In the assay for Recombinant Human PNMT Protein, CF (Catalog # 7854-MT) what is the purpose of the Hydrolysis Micture, Glutathione, and ThioGlo?
The assay described on the 7854-MTproduct insert is a coupled reaction. The first step, performed by PNMT, involves the transfer of a methyl group from norepinephrine to epinephrine. The second step involves conversion of s-adenylsylhomocysteine (a byproduct of the first reaction) to homocysteine, which has a detectable free thiol. This second step utilizes the Hydrolysis Mixture.
Glutathione also has a free thiol, so it can be used as a standard curve for the reaction.
ThioGlo is a thiol-reactive fluorescent probe. Upon reaction with a thiol group, a fluorescent adduct is formed that displays excitation/emission maxima of 380/445 nm, respectively.
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