Analysis Affinity and Kinetics of Rhodamine-Labeled C37 (R-C37) and 5-Helix by KinExA (CAT#: STEM-MB-0062-CJ)

Introduction

Infection by human immunodeficiency virus type 1 (HIV-1) involves the fusion of viral and cellular membranes mediated by formation of the gp41 trimer-of-hairpins. A designed protein, 5-Helix, targets the C-terminal region of the gp41 ectodomain, can disrupt trimer-of-hairpins formation and blocking viral entry. It is unclear at what point during fusion the gp41 HR2 segment becomes accessible to engineered inhibitors like 5-Helix. Unlike C-peptides, 5-Helix can precipitate Env from cellular and viral membranes in the absence of CD4 or coreceptor.

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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

Immunology/Inflammation;Virology

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: Lipids, Viruses, Serum
• Equipment: Kinetic Exclusion Assay (KinExA)

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