Recombinant Human CANT1 Protein, CF Summary
Gly80-Ile401, 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 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 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
- 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.
- 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.
- Dilute rhCANT1 to 0.005 µg/mL in Assay Buffer.
- Dilute Substrate to 100 µM in Assay Buffer.
- Load 50 µL of each dilution of the standard curve into a plate. Include a Curve Blank containing 50 μL of Assay Buffer.
- Load 25 µL of the 0.005 µg/mL rhCANT1 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
- Add 25 µL of 100 µM Substrate to the wells, excluding the standard curve and Curve Blank.
- Cover the plate with parafilm or a plate sealer and incubate at 37 °C for 20 minutes.
- Add 10 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
- Add 10 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
- Read plate at 620 nm (absorbance) in endpoint mode.
- 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
Background: Calcium Activated Nucleotidase 1/CANT1
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.
- Smith, T. et al. (2002) Arch. Biochim. Biophys. 406:105.
- Failer, B.U. et al. (2002) J. Biol. Chem. 277:36978.
- Huber, C. et al. (2009) Am. J. Hum. Genet. 85:706.
- Faden, M. et al. (2010) Am. J. Med. Genet. A. 152A:1157.
Product Specific NoticesCoomassie is a registered trademark of Imperial Chemical Industries Ltd.
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Phosphatase Activity Assays
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