>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
64 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
60-65 kDa, reducing conditions
Publications
Read Publications using 6246-GT in the following applications:
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
6 months from date of receipt, -20 to -70 °C as supplied.
3 months, -20 to -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Purity
>95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
Assay Buffer: 25 mM Tris, 150 mM NaCl, 10 mM MnCl2, 5 mM CaCl2, 150 mM K2SO4, pH 7.5
Recombinant C. difficile Toxin 1B/TcdB (rCdTcdB) (Catalog # 6246-GT)
Coupling Enzyme: Recombinant Human CD39L3/ENTPD3 (rhCD39L3) (Catalog # 4400-EN)
Substrate: UDP-Glucose (Calbiochem, Catalog # 670120), 10 mM stock in 25% ethanol
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 UDP-Glucose to 1.6 mM in Assay Buffer.
Dilute rhCD39L3 to 16 µg/mL in Assay Buffer.
Prepare reaction mixture by combining 200 μL UDP-Glucose and 200 μL rhCD39L3 (sufficient for ~15 wells).
Dilute rCdTcdB to 10 µg/mL in Assay Buffer.
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 (this is the first dilution of the standard curve).
Prepare six additional one-half serial dilutions of the 100 µM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5.0 nmol per well.
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 10 µg/mL rCdTcdB into the plate. Include a substrate blank containing 25 µL of Assay Buffer.
Add 25 µL of reaction mixture to the wells, excluding the standard curve.
Cover the plate with parafilm or a plate sealer and incubate at room temperature for 40 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix and incubate for 10 minutes at room temperature.
Add 100 µL of deionized water to all wells.
Add 30 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 Well:
rCdTcdB: 0.25 µg
rhCD39L3: 0.2 µg
Substrate: 400 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 C. difficile Toxin B/TcdB Protein, CF
TcdB
toxB
Background
Clostridium difficile is the leading cause of hospital-acquired diarrhea, known as C. difficile-associated disease. The estimated number of cases of C. difficile-associated disease exceeds 250,000 per year (1), with health care costs approaching US $1 billion annually (2). The major virulence factors produced by C. difficile are two toxins, TcdA and TcdB. Both toxins can monoglucosylate and inactivate Rho family small GTPases within target cells, leading to disruption of vital signaling pathways in the cell, subsequently causing diarrhea, inflammation, and damage of colonic mucosa (3, 4, 5). Both toxins have a similar tripartite structure comprised of an N‑terminal glucosyltransferase domain, a C-terminal receptor binding domain, and a small hydrophobic span possibly involved in toxin translocation (6). Our recombinant TcdB consists of the enzymatic domain. Both TcdA and TcdB also have potassium-dependent UDP-Glc hydrolase activity, which is essentially glucosyltransferase activity with water as the acceptor molecule (7). Under same conditions, UDP-glucose hydrolysis by TcdB occurs at a rate about 5-fold greater than that of TcdA.
Wilkins, T.D. and Lyerly, D.M. (2003) J. Clin. Microbiol 41:53.
Kyne, L. et al. (2002) Clin. Infect. Dis. 34:346.
Voth, D.E. and Ballard, J.D. (2005) Clin. Microbiol. Rev. 18:247.
Chaves-Olarte, E. et al. (1996) J. Biol. Chem. 271:6925.
Just I, et al. (1995) J. Biol. Chem. 270:13932.
Hammond, G.A. and Johnson, J.L. (1995) Microb. Pathog. 19:203.
Ciesla, W.P. Jr. and Bobak, D.A. (1998) J. Biol. Chem. 273:16021.
Publications for Toxin B/TcdB (6246-GT)(5)
We have publications tested in 3 confirmed species: Human, Mouse, Bacteria - Clostridium difficile.
We have publications tested in 1 application: Bioassay.
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