BA - Binding Assay

A binding assay is a type of experiment that is used extensively and is key in biological research. The assay measures the interaction between two molecules. For example, a protein binding with another protein, a small molecule, or nucleic acid, which is represented by an equilibrium dissociation constant (K_D).

There are two ways to measure affinity:

• Equilibrium experiment: Used to measure affinity by determining the extent of the reaction as a function of the concentration of one of the reactants.
• Kinetic experiment:Determined by the rates of forward and reserve reactions.

Types of binding assays based on light intensity:

• Absorbance-based binding assays
• Fluorescence-based binding assays
• Fluorescence polarization binding assays
• Time-resolved fluorescence (TRF) binding assays

Quantitative and qualitative detection in binding assays is important and because of this the target molecule or ligand molecule must be labelled. Labelling allows for light detection, which is commonly used.

Types of Radioligand assays:

• Saturation binding
• Scatchard plot
• Competition binding

Types of liquid phase assays:

• Immunoprecipitation
• ELISA

Types of solid phase assays:

• Multi-well plate
• On-bead binding
• Real-time cell-binding assays

General Protocol:

1. Prepare the target molecule by purifying it and immobilising it onto a solid support to facilitate the assay.
2. The ligand molecule needs to be labelled with a detectable signal.
3. Incubate the target and ligand molecule to allow binding.
4. Remove unbound ligands through washing.
5. Measure the bound molecules using a detection system.