Recombinant Human CANT1 Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human CANT1 Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze the beta 5'-phosphate group from the substrate uridine-5'-diphosphate (UDP). The orthophosphate product is measured by a Malachite Green Phosphate Detection Kit (Catalog # DY996). The specific activity is >85,000 pmol/min/μg, as measured under the described conditions.
• Source: Mouse myeloma cell line, NS0-derived human Calcium Activated Nucleotidase 1/CANT1 protein
  Gly80-Ile401, with an N-terminal 6-His tag
• Accession #: Q8WVQ1
• N-terminal Sequence: His
• Protein/Peptide Type: Recombinant Enzymes
• Gene: CANT1
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 37 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 36-40 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.
• Assay Procedure:
  • Assay Buffer: 50 mM MES, 5 mM CaCl₂, pH 6.5
  • Recombinant Human Calcium Activated Nucleotidase 1/CANT1 (rhCANT1) (Catalog # 6720-NT)
  • Substrate: Uridine 5’-diphosphate sodium salt (UDP) (Sigma, Catalog # U4125)
  • Malachite Green Phosphate Detection Kit (Catalog # DY996)
  • 96-well Clear Plate (Costar, Catalog # 92592)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute 1 M Phosphate Standard by adding 10 µL of the 1 M Phosphate Standard to 990 µL of deionized water for a 10 mM stock. Continue by adding 10 µL of the 10 mM Phosphate stock to 990 µL of Assay Buffer for a 100 µM stock.
  2. Prepare standard curve by performing seven one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.039 to 2.5 nmol per well.
  3. Dilute rhCANT1 to 0.005 µg/mL in Assay Buffer.
  4. Dilute Substrate to 100 µM in Assay Buffer.
  5. Load 50 µL of each dilution of the standard curve into a plate. Include a Curve Blank containing 50 μL of Assay Buffer.
  6. Load 25 µL of the 0.005 µg/mL rhCANT1 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
  7. Add 25 µL of 100 µM Substrate to the wells, excluding the standard curve and Curve Blank.
  8. Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
  9. Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
  10. Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
  11. Read plate at 620 nm (absorbance) in endpoint mode.
  12. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Phosphate released* (nmol) x (1000 pmol/nmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Substrate Blank.

  Per Reaction:
  • rhCANT1: 0.000125 µg
  • UDP: 50 µM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human CANT1 Protein, CF

• Apyrase homolog
• calcium activated nucleotidase 1
• CANT1
• DBQD
• EC 3.6.1.6
• Putative MAPK-activating protein PM09
• Putative NF-kappa-B-activating protein 107
• SCAN1
• SCAN-1Ca2+-dependent endoplasmic reticulum nucleoside diphosphatase
• SHAPY
• SHAPYmicromelic dwarfism with vertebral and metaphyseal abnormalities and advancedcarpotarsal ossification
• soluble Ca-activated nucleotidase, isozyme 1
• soluble calcium-activated nucleotidase 1
• soluble calcium-activated nucleotidase SCAN-1

Background

Calcium Activated Nucleotidase 1 (CANT1), is a Ca²⁺-dependent nucleoside diphosphatase which can hydrolyze a variety of nucleoside di- and triphosphates. CANT1 is also known as SCAN-1 and SHAPY. Its preferred substrates are UDP and GDP, and it is not active against nucleoside monophosphates (1). The CANT1 gene encodes a Type II membrane protein that is anchored in the endoplasmic reticulum (ER) membrane with its catalytic domain in the ER lumen (2). A secreted, soluble form of the enzyme also exists (1). CANT1 is expressed in many tissues, including fibroblasts and chondrocytes. Mutations of CANT1 are a cause of Desbuquois dysplasia, a condition characterized by deformation of bones and joints (2, 3). Recombinant human CANT1 was expressed as a secreted, soluble protein.

1. Smith, T. et al. (2002) Arch. Biochim. Biophys. 406:105.
2. Failer, B.U. et al. (2002) J. Biol. Chem. 277:36978.
3. Huber, C. et al. (2009) Am. J. Hum. Genet. 85:706.
4. Faden, M. et al. (2010) Am. J. Med. Genet. A. 152A:1157.

Bioinformatics

• Gene Symbol: CANT1
• Uniprot:
  • Q8WVQ1()