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Analysis Affinity of CoV-2 Virus Spike Protein and Membrane Receptor ACE-2 by KinExA (CAT#: STEM-MB-0019-CJ)

Introduction

The spike (S) glycoprotein on the surface of the virus particle of SARS-CoV-2 can bind to the host cell receptor, so that the virus membrane and the host cell fusion membrane occurs. The S protein of the virus is related to ACE2, and the binding of the S protein of SARS-CoV-2 to ACE2 may cause changes in the three-dimensional structure of the viral glycoprotein. This structural change can expose a cleavage site and increase viral susceptibility to cathepsin L cleavage, thereby enhancing viral fusion within the host cell. In addition, the S protein of SARS-CoV and SARS-CoV-2 consists of two functional regions called S1 and S2. Different subunits of the S1 domain are responsible for receptor binding and stabilization, while the S2 domain promotes structural rearrangement necessary for the fusion of virus and host cell membranes. For SARS-CoV and SARS-CoV-2, the S1 receptor binding domain (RBD) is required for these virions to bind to ACE2, thus contributing to the ability of these particles to cause host cell infections.




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

Virology;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: Serum, Plasma, Urine
• Equipment: Kinetic Exclusion Assay (KinExA)
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