Recombinant Human NAALADase-2 Protein, CF
Recombinant Human NAALADase-2 Protein, CF Summary
Product Specifications
Lys32-Leu740, 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.
7658-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 NaOH, 0.1% 2-mercaptoethanol (v/v)
- Recombinant Human NAALADase‑2/NAALAD2 (rhNAALAD2) (Catalog # 7658-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 (373 mM) stock in DMSO
- 16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: Gemini EM by Molecular Devices) or equivalent
- Dilute rhNAALAD2 to 0.5 µg/mL in Assay Buffer.
- Dilute Substrate to 40 µM in Assay Buffer.
- In reaction tube, mix 125 µL of rhNAALAD2 and 125 µL of Substrate. For a Control (no enzyme activity), deactivate 125 µL of rhNAALAD2 by heating it at 95-100 °C for 5 minutes, then add 125 µL of Substrate.
- Incubate the reaction tubes and Control at 37 °C for 60 minutes.
- Stop the reaction by heating at 95-100 °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 blank and transfer to the wells of a blank 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).
- rhNAALAD2: 0.025 µg
- Substrate: 10 µM
- o-PA: 7.5 mM
Reconstitution Calculator
Background: NAALADase-2/NAALAD2
NAALADase-2 (N-acetylated-alpha-linked acid dipeptidase 2) is a Type II integral membrane protein. This zinc metallopeptidase is also known as glutamate carboxypeptidase III (1). It is a member of the transferrin receptor family of proteins and is structurally related to the Prostate Specific Membrane Antigen (PSMA), also known as glutamate carboxypeptidase II and NAALADase-1 (2, 3). Like PSMA, NAALADase-2 cleaves N-acetyl-Asp-Glu to release free glutamate (1). However, the two enzymes differ in their expression patterns (3). NAALADase-2 is most highly expressed in testis and the pituitary gland, while PSMA expression is greatest in the nervous system, prostate, kidney, and small intestine. Recombinant human NAALADase-2 was expressed with a signal sequence instead of its N-terminal signal‑anchor domain, resulting in its secretion.
- Hlouchova, K. et al. (2007) J. Neurochem. 101:682.
- Hlouchova, K. et al. (2009) FEBS J. 276:4448.
- Hlouchova, K. et al. (2012) Curr. Med. Chem. 19:1316.
Product Specific Notices
Coomassie is a registered trademark of Imperial Chemical Industries Ltd.FAQs
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