Recombinant Botulinum Neurotoxin Type F Light Chain, CF

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

Recombinant Botulinum Neurotoxin Type F Light Chain, CF Summary

• Details of Functionality: Measured by the cleavage of the substrate GFPuv/SNAP/VAMP in a gel-shift assay. >40% of 1 μg substrate is cleaved by 50 ng, as measured under the described conditions.
• Source: E. coli-derived c. botulinum BoNT-F Light Chain protein
  Pro2-Gly420, with an N-terminal Met and 6-His tag
• Accession #: P30996
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >90%, 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: 49 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: 48-52 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, Tween® 20 and Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 0.05% Tween® 20, pH 7.5
  • Recombinant C. botulinum BoNT‑F Light Chain (rBoNT/F-LC) (Catalog # 7380-ZN)
  • Substrate: Recombinant GFP/SNAP25B/VAMP-2 (Catalog # 7375-SV)
  • SDS-PAGE and silver stain reagents or equivalent or Western blot with appropriate antibodies
  1. Dilute Substrate to 100 µg/mL in Assay Buffer.
  2. Dilute rBoNT/F-LC to 5 µg/mL in Assay Buffer.
  3. Combine equal volumes of diluted Substrate with diluted rBoNT/F-LC. Prepare two controls by combining equal volumes of diluted Substrate with Assay Buffer.
  4. Incubate reaction vials at 37 °C for 1 hour. Incubate one control at 37 °C and the other at -20 °C for 1 hour.
  5. After incubation, combine reaction mixtures and controls with reducing SDS-PAGE sample buffer at a 1:1 (reaction mixture:sample buffer) ratio (v/v) to stop reactions.
  6. Analyze the cleavage products by SDS-PAGE (Load 40 µL of the mixture from step 5 per lane, 1 µg Substrate per lane) followed by silver staining and/or Western blot.
  Per Lane:
  • rBoNT/F-LC: 50 ng
  • Substrate: 1 µg

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd. Tween is a registered trademark of ICI Americas.

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

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

• BoNTF Light Chain
• BoNT-F Light Chain

Background

Botulinum Neurotoxin Type F 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 that are activated by proteolytic cleavage to create the light and heavy chains that are linked by a disulfide bond. The 50 kDa light chain contains the metalloprotease domain whereas the 100 kDa heavy chain contains a receptor binding domain and a domain required for translocation across the cell membrane (3).  BoNTs are the most toxic protein toxins known for humans.  As zinc proteases, they cleave SNARE proteins to elicit flaccid paralysis in botulism poisoning. Cleavage of the SNARE proteins results in the blocking of acetylcholine release at the neuromuscular junction (2‑4). E. coli expressed recombinant light chains are active proteases. In the absence of heavy chains, however, they lack toxicity because they cannot enter into host cells.

1. Campbell, K.D. et al. (1993) J. Clin. Microbiol. 31:2255.
2. Montecucco, C. and S. Giampietro (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

• Uniprot:
  • P30996(C. botulinum)