Purity
>95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to cleave the fluorogenic peptide substrate, VAMPtide. The specific activity is >20 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .
Source
E. coli-derived Pro2-His428, with an N-terminal Met and 6-His tag
Accession #
N-terminal Sequence
AnalysisMet
Predicted Molecular Mass
50 kDa
SDS-PAGE
47 kDa, reducing conditions
5420-ZN |
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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.
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Assay Procedure
Materials
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).
Per Well:
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.
References:
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.
Long Name:
Botulinum Neurotoxin Type B Light Chain
Entrez Gene IDs:
6030102 (C. botulinum)
Alternate Names:
BoNTB Light Chain; BoNT-B Light Chain
