Recombinant Botulinum Neurotoxin Type B Light Chain, CF Summary
Pro2-His428, with an N-terminal Met and 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, NaCl, Tween® 20 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, 0.05% (v/v) Tween® 20, pH 6.5
- Recombinant C. botulinum BoNT-B Light Chain (rBoNT/B-LC) (Catalog # 5420-ZN)
- Fluorogenic Substrate: VAMPtide (o-Abz/Dnp) (List Biological Laboratories, Inc., Catalog # 540), 2 mM in DMSO
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute rBoNT/B-LC to 10 µg/mL in Assay Buffer.
- Dilute fluorogenic substrate VAMPtide to 20 µM in Assay Buffer.
- Combine equal volumes of 10 µg/mL rBoNT/B-LC and 20 µM Substrate in reaction tubes. Incubate at 37 °C for 20 minutes.
- Load into a black well plate 100 µL of the reaction mixture.
- Read at excitation and emission wavelengths of 320 nm and 410 nm (top read), respectively, in endpoint mode.
- Calculate specific activity:
Specific Activity (pmoles/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 Abz-Gly (Bachem, Catalog # E-2920).
- rBoNT/B-LC: 0.50 µg
- Substrate: 10 µM
Background: BoNT-B Light Chain
Botulinum Neurotoxin Type B is one of the seven serotypes of Botulinum Neurotoxins (BoNTs) produced by various strains of Clostridium botulinum (1, 2). BoNTs are synthesized as inactive single chain protein precursors and activated by proteolytic cleavage to generate disulfide-linked two-chain proteins. The 50 kDa light chain contains the catalytic domain, whereas the 100 kDa heavy chain contains an internal translocation domain and a receptor binding domain (3). BoNTs are the most potent protein toxins for humans. As zinc proteases, they cleave SNARE proteins to elicit flaccid paralysis in botulism by blocking acetylcholine release at the neuromuscular junction (2-4). E. coli expressed recombinant light chains are active proteases. In the absence of the heavy chains, however, they lack toxicity because they cannot enter into host cells.
- Campbell K.D. et al. (1993) J. Clin. Microbiol. 31:2255.
- Montecucco, C. and Giampietro, S. (1993) Trends Biochem. Sci. 18:324.
- Turton, K. et al. (2002) Trends Biochem. Sci. 27:552.
- Schiavo, G. et al. (2000) Physiol. Rev. 80:717.
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