Recombinant Botulinum Neurotoxin Type A Light Chain, CF

• Reactivity: Ba-Cb
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Botulinum Neurotoxin Type A Light Chain, CF Summary

• Details of Functionality: Measured by its ability to cleave the fluorogenic peptide substrate, SNAPtide. The specific activity is >1 pmol/min/µg, as measured under the described conditions.
• Source: E. coli-derived c. botulinum BoNT-A Light Chain protein
  Met1-Phe425, with an N-terminal Met and 6-His tag
• Accession #: Q45894
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: atx
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Endotoxin Note: <1.1 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 50 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: 52 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: 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, NaCl and Tween® 20.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by silver stain
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 0.05% (v/v) Tween® 20, pH 7.5
  • Recombinant C. botulinum BoNT‑A Light Chain (rBoNT/A-LC) (Catalog # 4489-ZN)
  • Substrate: SNAPtide (FITC/DABCYL) (List Biological Laboratories, Inc., Catalog # 521), 2 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: Spectra Max Gemini EM by Molecular Devices) or equivalent
  1. Dilute rBoNT/A-LC to 10 µg/mL in Assay Buffer.
  2. Dilute Substrate to 10 µM in Assay Buffer.
  3. Load into a black well plate 50 µL of 10 µg/mL of rBoNT/A-LC and start the reaction by adding 50 µL of 10 µM Substrate. As a Substrate Blank combine 50 µL of 10 µM Substrate and 50 µL of Assay Buffer.
  4. Read at excitation and emission wavelengths of 490 nm and 523 nm (top read), respectively, in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)) / (amount of enzyme (µg))

  *Adjusted for Substrate Blank
  **Derived using calibration standard Unquenched Calibration Peptide (List Biological Laboratories, Catalog # 528).

  Per Well:
  • rBoNT/A-LC: 0.50 µg
  • Substrate: 5 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Botulinum Neurotoxin Type A Light Chain, CF

• BoNTA Light Chain
• BoNT-A Light Chain

Background

Botulinum neurotoxin type A 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. However, they are not toxic because they cannot enter into host cells in the absence of the heavy chains.

1. Willems, A. et al. (1993) Res. Microbiol. 144:547.
2. Montecucco, C. and Giampietro, S. (1993) Trends. Biochem. Sci. 18:324.
3. Turton, K., et al. (2002) Trends. Biochem. Sci. 27:552.
4. Schiavo, G. et al. (2000) Physiol. Rev. 80:717.

Bioinformatics

• Gene Symbol: atx
• Uniprot:
  • Q45894(C. botulinum)