Botulinum Neurotoxin Type B Light Chain, Recombinant

Materials:

...Botulinum toxin-B List biological Cat# 620A ...

• Product #620A - Botulinum Neurotoxin Type B Light Chain, Recombinant

Reagents:

... BoNT/B light chain were purchased from List Biological Laboratories (Campbell, CA, USA). ...

Assay for BoNT Endopeptidase Activity:

The cleavage of the recombinant GST-Synaptobrevin-II protein substrate by the BoNT/B light chain domain was performed as suggested by the manufacturer (List Biological Laboratories Inc., Campbell, CA, USA) with minor modifications. Briefly, 10 M of GST-Synaptobrevin-II protein substrate was incubated with or without 60 nM of BoNT/B light chain and were then were treated with 2.5 g individual mAbs or a combination of antibodies for 3 h at 37 C (control samples were not pre-treated with mAbs) in a 10 L reaction (20 mM HEPES, pH 7.4, 1.25 mM DTT, 0.3 mM ZnCl₂ and 0.2% Tween-20). ...

• Product #620A - Botulinum Neurotoxin Type B Light Chain, Recombinant

Clostridial neurotoxin preparation for Western blotting:

VAMP-specific tetanus neurotoxin light-chain (TeNT-LC) and botulinum neurotoxins B and D light-chain (BoNT/B-LC and BoNT/D-LC, respectively) were purchased from List Biologicals Laboratories, Inc. (Campbell, CA). Control neurotoxin solutions were placed in a boiling water bath for 30 min to denature and inactivate the neurotoxins.

Clostridial neurotoxin preparation for pressure injection:

The lyophilized form of TeNT-LC and BoNT/B/D-LC was reconstituted with a solution consisting of 100 mM KCl and 300 M 3 kDa neutral dextran-Texas red (Life Technologies Corp., Carlsbad, CA) such that the concentration of TeNT-LC and BoNT/B-LC were 0.5 g/L and BoNT/D-LC was 0.3 g/L. The fluorescent dextran-Texas red dye was used to allow visualization of neurotoxin injection. Control neurotoxin solutions were denatured in a boiling water bath for 30 min.

In vitro BoNT LC metalloprotease activity:

Determination of BoNT/A LC percent inhibition by SMNPIs was performed as previously described.  For BoNT/B LC percent inhibition, the HTS-assay utilizes a commercially available fluorogenic substrate from List Biological Laboratories (Campbell, CA). Briefly, a multichannel pipette was used to add fluorescent substrate (final concentration 20 M) and small molecules (final concentration 20 M) to a 96 well microplate. The reactions were initiated by adding recombinant BoNT/B LC (final concentration 40 nM) to each well. All reactions were conducted in a buffered solution consisting of 40 mM Hepes pH 7.2, 1 mM DTT and 100 M ZnCl2. The change in fluorescence intensity over time was continuously monitored in each well using a 96 well plate fluorimeter (Saphire 2, Tecan, Mnnedorf, Switzerland). The assays were run at 37 C, quenched by the addition of TFA, and analyzed using reverse-phase HPLC. Potential SMNPIs of the BoNT B LC were identified by comparing the reaction velocities (change in fluorescence intensity over time) of samples assayed in the presence of compounds versus  control samples.

Author did not specify which List Biological Laboratories product was utilized.  Refer to the follwing List Labs link for Botulinum products available:  https://listlabs.com/product-information/botulinum-toxins/