Recombinant Cynomolgus PSMA/FOLH1/NAALADase I Protein, CF Summary
with an N-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 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 Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.5
- o-PA Buffer: 0.2 M NaOH containing 0.1% 2-mercaptoethanol (v/v)
- Recombinant Cynomolgus Monkey PSMA/FOLH1/NAALADase I His-tag (rcynoPSMA) (Catalog # 10437-ZN)
- Substrate: Ac-Asp-Glu (Sigma, Catalog # A5930), 10 mM stock in 40 mM NaOH
- o-pthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rcynoPSMA to 0.4 µg/mL in Assay Buffer.
- Dilute Substrate to 40 µM in Assay Buffer.
- Combine 125 µL of 0.4 µg/mL rcynoPSMA and 125 µL of 40 µM Substrate. For a Control, inactivate 125 µL of 0.4 µg/mL
rcynoPSMA by heating it at 95 °C for 5 minutes, then add 125 µL of 40 µM Substrate.
- Incubate the reaction tubes and control tubes at 37 °C for 60 minutes.
- Stop the reaction by heating all tubes 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 control. Vortex and incubate at room temperature for 10 minutes.
- Load 200 µL of each reaction and control 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 Control
**Derived using calibration standard L-Glutamic Acid (Sigma, Catalog # G8415)
- rcynoPSMA: 0.020 µg
- Substrate: 10 µM
- o-PA: 7.5 mM
2 μg/lane of Recombinant Cynomolgus Monkey PSMA/FOLH1/NAALADase I His-tag (Catalog # 10437-ZN) was resolved with SDS-PAGE under reducing (R) and non-reducing (NR) conditions and visualized by Coomassie® Blue staining, showing a band at ~100 kDa under reducing conditions.
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). PMSA has a short cytosolic N-terminal domain, a single membrane-spanning segment, and an extracellular region that is composed of a protease domain, apical domain, and C-terminal domain (3). The extracellular domains all contribute to substrate recognition. The protein forms an active homodimer reliant on interactions between amino-acid side chains and glycosylation (3,4). PSMA is also known as glutamate carboxypeptidase II (GCPII), folate hydrolase 1, and N-acetylated-alpha-linked acidic dipeptidase-1 (NAALADase1). PSMA activity plays a role in tumor angiogenesis making it not only a tumor marker, but a therapeutic target in cancers including prostate cancer (5). In the brain, PSMA hydrolyzes the neurotransmitter N-acetyl-Asp-Glu (NAAG) 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, schizophrenia, Alzheimer's, and amyotrophic lateral sclerosis (6,7,8). Intestinal PSMA hydrolyzes folylpoly-gamma -glutamates, facilitating the uptake of folate (8). Upregulation of PSMA is present in inflammatory bowel disease, Crohn's disease, and ulcerative colitis where pharmacological inhibition has shown amelioration of clinical symptoms pertaining to these diseases in mice (5).
- Silver, D.A. et al. (1997) Clin. Cancer Res. 3:81.
- Carter, R.E. et al. (1996) Pro. Natl. Acad. Sci. USA. 93:749.
- Mesters, J.R. et al. (2006) EMBO J. 25:1375.
- Shulke, N. et al. (2003) Proc. Natl. Acad. Sci. USA. 100:12590.
- Vornov, J.J. et al. (2019) Neurochem. Res. [Epub ahead of print].
- Jackson, P.F. and Slusher, B.S. (2001) Curr. Med. Chem. 8:949.
- Neale, J.J. and T. Yamamoto. (2020) Prog. Neurobiol. 184:101722.
- Heston, W.D. (1997) Urology 49:104.
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