Analysis Affinity of Protein–Protein Interactions by KinExA (CAT#: STEM-MB-0052-CJ)

Introduction

Proteins, as ligands, signaling partners, or messengers, form vast networks that require specific interactions to carry out their functions. Generally, these binding events can be thought of as the propensity of two protein-binding partners to either associate, or conversely dissociate at different concentrations. Protein partners that come together rapidly in solution at low relative concentrations, and stay bound together for an extended period of time are thought to have a low propensity for dissociation, and are thus thought of as stronger binders.

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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, Toxicology

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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