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Analysis Biomolecular Interactions of BoNT/B to Syt-II40-60 by BLI (CAT#: STEM-MB-0149-CJ)

Introduction

Botulinum neurotoxins (BoNTs) are bacterial proteins that cause the life-threatening disease botulism, and are considered as among the highest-risk threat agents for bioterrorism (the "Class A agents"). Seven antigenically-distinct BoNT serotypes (designated A to G) are produced by several species of anaerobic Clostridia: C. botulinum, C butyricum, C. baratii, and C. argentinense. Botulism is a disease with four distinct, naturally-occurring syndromes: foodborne, wound, infant botulism, and adult intestinal toxemia. Inhalational botulism can result from aerosolization of the toxin. All BoNT serotypes act via similar mechanisms on their target nerve cell: initial binding of the C-terminal portion of the heavy chain through ganglioside and protein receptors on the presynaptic cell surface, followed by internalization into and translocation within the nerve ending by the N-terminal portion of the heavy chain. Each BoNT serotype has specific action site. Serotypes B, D, F, and G cleave vesicle associated membrane protein (VAMP or synaptobrevin).




Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Immunology/Inflammation; Virology; Pharmacology

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Streptavidin Magnetic Beads