Recombinant Human CNDP2/CPGL Protein, CF
Discontinued Product
Recombinant Human CNDP2/CPGL Protein, CF Summary
Product Specifications
Met1-Asp475, with a C-terminal 10-His tag
Analysis
Ser299 (C-terminal fragment) & Ala2 predicted (full length & N-terminal fragment)
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.
3560-ZN
| Formulation | Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol. |
| Shipping | The product is shipped with dry ice or equivalent. 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 Tris, 0.1 mM MnCl2, pH 9.0
- Recombinant Human Cytosol Nonspecific Dipeptidase (CNDP2)/CPGL (rhCNDP2) (Catalog # 3560-ZN)
- Substrate: L-Carnosine ( beta -Ala-His) (Sigma, Catalog # C9625)
- o-Phthaldialdehyde (o-PA) (Sigma, Catalog # P0657), 50 mg/mL stock in DMSO
- NaOH, 2 M stock in deionized water
- Trichloroacetic Acid (TCA) (Sigma, Catalog # T6399), 10% stock in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rhCNDP2 to 10 ng/μL in Assay Buffer.
- Dilute Substrate to 2 mM in Assay Buffer.
- Combine 75 μL of 10 ng/μL rhCNDP2 and 75 μL of Substrate. Include a Blank containing 75 μL of rhCNDP2 only.
- Incubate for 1 hour at room temperature. Protect from light.
- Stop the reaction by adding 75 μL of 1% TCA. Also add 75 μL of 1% TCA to the Blank. Mix well.
- Now add 75 μL of 2 mM Substrate to the Blank after the TCA addition.
- Centrifuge all vials at 13,000 rpm in a microfuge for 10 minutes.
- Transfer 180 μL of supernatant to new tubes.
- Dilute o-PA to 5 mg/mL in 2 M NaOH.
- Add 60 μL of 5 mg/mL o-PA to each tube.
- Vortex well and incubate at room temperature for 30 minutes. Protect from light.
- Load 200 μL of the reaction mixture into a plate.
- Read at excitation and emission wavelengths of 360 nm and 460 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-Histidine (Sigma, Catalog # H-6034).
- rhCNDP2: 0.500 μg
- o-PA: 1.25 mg/mL
- Substrate: 0.5 mM
Reconstitution Calculator
Background: Cytosol Nonspecific Dipeptidase (CNDP2)/CPGL
The human CNDP2 gene encodes cytosol non-specific dipeptidase, a member of the M20 family of metalloproteases (1, 2). Also known as glutamate carboxypeptidase-like protein 1 (CPGL), tissue carnosinase or peptidase A, CNDP2 is ubiquitously expressed throughout human tissues. Unlike CNDP1, CNDP2 has broad substrate specificity for dipeptides with free amino and carboxyl groups. The purified recombinant human CNDP2 migrates as three bands in SDS-PAGE under reducing conditions that correspond to the full length protein (54 kDa), the N-terminal fragment (33 kDa) and the C-terminal fragment (22 kDa). The initiator Met1 may be removed and Ala2 may be acetylated based on Accession # Q96KP4.
- Teufel, M. et al. (2003) J. Biol. Chem. 278:6521.
- Woessner, J. (2004) in Handbook of Proteolytic Enzymes (ed. Barrett, A.J. et al.) pp.1020, Academic Press, San Diego.
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