Recombinant C. difficile Toxin B/TcdB Protein, CF Summary
Met1-Leu543, with a C-terminal 6-His tag
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 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 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
Background: Toxin B/TcdB
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.
Citations for Recombinant C. difficile Toxin B/TcdB Protein, CF
R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.
Citations: Showing 1 - 3
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Investigation of the adsorption capacity of the enterosorbent Enterosgel for a range of bacterial toxins, bile acids and pharmaceutical drugs
Authors: CA Howell, SV Mikhalovsk, EN Markaryan, AV Khovanov
Sci Rep, 2019;9(1):5629.
Species: Bacteria Toxins
Sample Types: Gel Constructs
Treatment ofClostridium difficileinfection with a small molecule inhibitor of toxin UDP-glucose hydrolysis activity
Authors: IL Stroke, JJ Letourneau, TE Miller, Y Xu, I Pechik, DR Savoly, L Ma, LJ Sturzenbec, J Sabalski, PD Stein, ML Webb, DW Hilbert
Antimicrob. Agents Chemother., 2018;0(0):.
Species: Bacteria - Clostridium difficile
Sample Types: Chemical Compound
Pyrin inflammasome activation and RhoA signaling in the autoinflammatory diseases FMF and HIDS
Authors: Yong Hwan Park
Nat Immunol, 2016;0(0):.
Sample Types: Whole Cells
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Phosphatase Activity Assays
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