Affinity Electrophoresis

Affinity electrophoresis (AEP) is a helpful technique for separating biomolecules, such as nucleic acids, plasma proteins, enzymes, receptors, lectins, and extracellular matrix proteins in electric fields through specific interactions with their ligands and determining dissociation constants for those interactions. 

Two-dimensional affinities electrophoresis (2-D AEP), a recently developed technique that combines isoelectric focussing with AEP, has been used to investigate the immunological response to haptens.

Anti-hapten antibodies were isolated by 2-D AEP from a significant number of groups of IgG spots using a few microliters of antiserum after a mouse was immunised with hapten-conjugated bovine serum albumin.

Each set of spots had the same affinity for the hapten but varied isoelectric points, as in the case of hapten-specific monoclonal antibodies.

This allowed us to investigate the diversity and suitable stabilisation of anti-hapten antibodies throughout the course of a single animal vaccination.

Furthermore, the effect of a carrier and hapten arrays on the generation of anti-hapten antibodies, as well as the source of charge heterogeneity in monoclonal antibodies, were investigated to better understand the molecular mechanism of the immune response in vivo.

Capillary affinity electrophoresis (CAE) is a technique that combines capillary electrophoresis with the utilisation of molecular forces in free solutions. As affinity probes in CAE, fluorophore-labelled compounds with affinity for the target molecules are utilised.

Capillary electrophoresis is used to separate the complexes formed by mixing the affinity probes and samples. The approach is based on the idea that intermolecular interactions modify the mobility of a target molecule. A detection method based on scanning laser-induced fluorescence allows for detection with great sensitivity and precision, even with small volumes of material (1 L). This section focuses on advancements in CAE approaches that include fluorescence tagging of affinity ligands.

Affinity Electrophoresis Theory

Affinity electrophoresis is a type of zone gel electrophoresis that involves proteins binding to ligands (specific macromolecules). As a result, affinity electrophoresis and immunoelectrophoresis (see above) are referred to as ligand electrophoresis. Lentils, enzyme substrates, and other compounds serve as ligands. These are not attached to agarose or polyacrylamide gel, as opposed to affinity chromatography ligands, which are fixed on a matrix. The ligands bind to and form a complex with the appropriate polyions.

Additional electric charges and, as a result, other mobilities are present in the complexes.

Affinity electrophoresis techniques include lectin electrophoresis, affinity supported molecular matrix electrophoresis, charge shift electrophoresis, affinity-trap electrophoresis, saccharide affinity capillary, affinity electrophoresis, and mobility shift electrophoresis.

Electrophoresis of Phosphate Affinity

Phosphate affinity electrophoresis employs an affinity probe, known as a ‘Phos-Tag’, which consists of a molecule that binds exclusively to ionic phosphate ions in neutral aqueous.

A separation gel consisting of an acrylamide-dependent Phos-Tag monomer that has been copolymerised is also used in this procedure.

Compared to non-phosphorylated proteins, phosphorylated proteins move more slowly through the gel. This approach allows the researcher to see changes in the phosphorylation levels of any particular protein.

Method of Separation

Separation-based approaches physically separate the free and bound solutes and calculate their concentrations. They are further categorised as heterogeneous or homogeneous based on how the separation is performed. Chromatography, dialysis, ultrafiltration, and surface plasmon resonance (SPR) are all different types of procedures. On the surface of a solid substrate, the free solute is separated from the bound one. The homogenous approaches include affinities capillary electrophoresis (ACE) and electrospray ionisation mass spectrometry (ESI-MS). The separation might take place in solutions or in the gas phase.

Method without Separation

Non-separation approaches track how certain physicochemical characteristics of the solute or ligand change during complexation.

Spectroscopy (e.g., infrared (IR), ultraviolet-visible (UV-Vis), nuclear magnetic resonance (NMR), fluorescence (e.g., electrochemical techniques potentiometry, conductimetry, and polarography), and phase-solubility and hydrolysis kinetics are also included in this area.

Conclusion

Affinity Capillary Electrophoresis uses the benefits of capillary electrophoresis for the investigation of protein interactions. ACE is favourable due to its excellent separation efficiency, quicker analysis time, ability to run at physiological pH, and low ligand/molecule consumption. Furthermore, the structure of the protein is not required to conduct ACE experiments. The fundamental disadvantage is that it does not provide much stoichiometric data about the reaction under investigation.