Analysis Affinity and Kinetics of BoNT/H by KinExA (CAT#: STEM-MB-0057-CJ)

Introduction

Botulinum neurotoxin (BoNT) is the most poisonous substance known and has 3 functional domains: a binding domain (HC) , a translocation domain (HN) , and a catalytic domain (LC). The HC binds receptors on the presynaptic membrane, leading to BoNT endocytosis.In addition,BoNT also causes botulism in humans, is a widely used therapeutic, and is also a Tier 1 (mass casualty capable) potential bioweapon. For these reasons, antitoxins neutralizing all BoNT serotypes are needed. A heptavalent (targeting serotypes A–G) equine botulism antitoxin (BAT) produced from immunized horses is licensed in the United States for the treatment of botulism. BoNT/H is produced by bivalent C. botulinum strain IBCA10-7060 that also produces BoNT/B2. Compared with the other 7 BoNT serotypes, BoNT/H has an N-terminal two-thirds that is most homologous to BoNT/F and a C-terminal one-third that is most homologous to BoNT/A.

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Principle Applications Procedure Materials Notes Related Products

Principle

KinExA is a two-stage analysis system. In the first stage, a number of solutions are prepared, where one partner remains constant (constant binding partner, or CBP) and the other (titrant) is variable, usually serially diluted. As the titrant is added, the free CBP decreases and is analysed by a sophisticated and precise microfluorescence measurement device. The signal generated can be mathematically related to the affinity (KD) of the two molecules for each other, as well as the kinetic parameters of binding (kon) and dissociation (koff).

Applications

Oncology & Cancer; Immunology/Inflammation; Pharmacology

Procedure

1. preparation of the functionalized beads which will capture the analyte for measuremen.
2. preparation of a series of solutions consisting of a constant initial concentration of one component of the binary reaction and serial dilutions of the other reactant. The component that is kept constant is the constant binding partner (CBP) , and is the one which will be analyzed.
3. each reaction mixture is sampled and the fluorescence of free CBP bound to the capture beads is obtained for subsequent numerical analysis.

Materials

• Sample Type: Antibodies, Cells, Small Molecules, Proteins, DNA, Lipids, Viruses, Serum, & More
• Equipment: Kinetic Exclusion Assay (KinExA)

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