Recombinant Mouse PSMA/FOLH1 Protein, CF Summary
Product Specifications
Ile44-Ala752, with an N-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.
4946-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in MES 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: 50 mM HEPES, 0.1 M NaCl, pH 7.5
- o-PA Buffer: 0.2 M Sodium Hydroxide containing 0.1% mercaptoethanol (v/v)
- Recombinant Mouse PSMA/FOLH1/NAALADase I (rmNAALADase I) (Catalog # 4946-ZN)
- Substrate: Ac-Asp-Glu (Sigma, Catalog # A5930), 10 mM stock in 40 mM Sodium Hydroxide
- o-phthaldialdehyde (o-PA) (Sigma, Catalog # P0657) - 50 mg/mL (0.373 M) stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rmNAALADase I to 0.2 µg/mL in Assay Buffer.
- Dilute Substrate to 40 µM with Assay Buffer.
- In reaction tube, mix 125 µL of rmNAALADase-1 and 125 µL of Substrate. For a Substrate Blank (no enzyme activity control), deactivate 125 µL of rmNAALADase-1 by heating it at 95 °C for 5 minutes, then add 125 µL of Substrate.
- Incubate the reaction tubes and blank control at 37 °C for 60 minutes.
- Stop the reaction tubes by heating at 95 °C for 5 minutes, then cool to room temperature.
- Prepare a 15 mM o-PA solution in o-PA Buffer.
- Add 250 µL of o-PA solution to each reaction and blank. Vortex and incubate at room temperature for 10 minutes.
- Remove two 200 µL aliquots from each reaction and control and transfer to the wells of a black microplate.
- Read at excitation and emission wavelengths of 330 nm and 450 nm (top read), respectively, in endpoint mode.
- Calculate specific activity:
|
Specific Activity (pmol/min/µg) = |
Adjusted Fluorescence* (RFU) x Conversion Factor** (pmol/RFU) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415).
- rmNAALADase I: 0.010 µg
- Substrate: 10 µM
- o-PA: 7.5 mM
Reconstitution Calculator
Background: PSMA/FOLH1/NAALADase I
Prostate-specific membrane antigen (PSMA), a tumor marker in prostate cancer encoded by the FOLH1 gene, is a type II transmembrane zinc metallopeptidase that is most highly expressed in the nervous system, prostate, kidney, and small intestine (1, 2). The enzyme is also known as glutamate carboxypeptidase II (GCPII), folate hydrolase 1, folylpoly-(-glutamate carboxypeptidase (FGCP), and N-acetylated-alpha-linked acidic dipeptidase-1 (NAALADase1). In the brain, PSMA hydrolyzes the neurotransmitter N-acetyl-Asp-Glu to produce glutamate, another neurotransmitter. Inhibition of brain PSMA activity is considered to be a promising approach for the treatment of neurological disorders associated with glutamate excitotoxicity, such as stroke, chronic pain, and amyotrophic lateral sclerosis (3). Intestinal PSMA hydrolyzes folylpoly-gamma -glutamates, facilitating the uptake of folate (4).
- Silver, D.A. et al. (1997) Clin. Cancer Res. 3:81.
- Carter, R.E. et al. (1996) Pro. Natl. Acad. Sci. USA. 93:749.
- Jackson, P.F. and Slusher, B.S. (2001) Curr. Med. Chem. 8:949.
- Heston, W.D. (1997) Urology 49:104.
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