Recombinant Botulinum Neurotoxin Type A Light Chain, CF

R&D Systems | Catalog # 4489-ZN

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Botulinum Neurotoxin Type A Light Chain (4489-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

Q45894

Applications

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

Product Specifications

Source

E. coli-derived c. botulinum BoNT-A Light Chain protein
Met1-Phe425, with an N-terminal Met and 6-His tag

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by silver stain.

Endotoxin Level

<1.1 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

Met

Predicted Molecular Mass

50 kDa

SDS-PAGE

52 kDa, reducing conditions

Activity

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.

Formulation, Preparation, and Storage

4489-ZN
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Tween® 20.
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-A Light Chain

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.

References

  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.

Long Name

Botulinum Neurotoxin Type A Light Chain

Alternate Names

BoNTA Light Chain

Entrez Gene IDs

5185061 (C. botulinum)

Gene Symbol

BONT

UniProt

Q45894 (C. botulinum)

Additional BoNT-A Light Chain Products

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

For research use only

Related Research Areas

Metalloproteases and Regulators

Citations for Recombinant Botulinum Neurotoxin Type A Light Chain, CF

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Protocols

View specific protocols for Recombinant Botulinum Neurotoxin Type A Light Chain, CF (4489-ZN):

Materials
  • 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

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