Recombinant Botulinum Neurotoxin Type D Light Chain, CF

R&D Systems | Catalog # 6037-ZN

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Botulinum Neurotoxin Type D Light Chain (6037-ZN)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P19321

Applications

Enzyme Activity
View all BoNT-D Light Chain Proteins and Enzymes »

Product Specifications

Source

E. coli-derived c. botulinum BoNT-D Light Chain protein
Thr2-Ser428, with an N-terminal Met and 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<1.0 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

50 kDa

SDS-PAGE

41-45 kDa, reducing conditions

Activity

Measured by the cleavage of the substrate GFPuv/SNAP/VAMP in a gel-shift assay.
>50% of 1 μg substrate is cleaved by 4 ng, as measured under the decribed conditions.

Formulation, Preparation, and Storage

6037-ZN
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl, Tween® and Glycerol.
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.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.

Background: BoNT-D Light Chain

Botulinum Neurotoxin Type D 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.

References

  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.

Long Name

Botulinum Neurotoxin Type D Light Chain

Alternate Names

BoNTD Light Chain

UniProt

P19321 (C. botulinum)

Additional BoNT-D Light Chain Products

Product Documents for Recombinant Botulinum Neurotoxin Type D Light Chain, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Botulinum Neurotoxin Type D Light Chain, CF

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

For research use only

Related Research Areas

Metalloproteases and Regulators

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Protocols

View specific protocols for Recombinant Botulinum Neurotoxin Type D Light Chain, CF (6037-ZN):

Materials
  • Assay Buffer: 50 mM HEPES, pH 6.5
  • Recombinant C. botulinum BoNT‑D Light Chain (rBoNT/D-LC) (Catalog # 6037-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/D-LC to 0.4 µg/mL in Assay Buffer.
  3. Combine equal volumes of diluted Substrate with diluted rBoNT/D-LC. Prepare two controls by combining equal volumes of diluted Substrate with Assay Buffer.
  4. Incubate reaction vials at room temperature for 1 hour. Incubate one control at room temperature 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/D-LC: 4 ng
  • Substrate: 1 µg

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