What Is Affinity Chromatography Using The Natural Properties Of Protein

Affinity Chromatography using the natural properties of protein

Introduction

The motivation for developing affinity chromatography is clear and simple, it is because protein has to purify before research studied its structure and mechanism of its’ functions, and each protein has various sizes, mass, pH value, and solubility. However, it is a difficult to separate target protein through single method in “five decades ago” .1 During the early time, scientist used less efficient method to separate protein base on pH, ionic strength and temperature. Later a more advanced and efficient method, affinity chromatography (AC), have been invented, and replaced the previous method, and used widely overall the laboratory. German scientist, Emil starkenstein,

When AC is used to purify and separate large biomolecule from a complex mixture, those techniques aspects have to be consider. The main function of matrix is allowed ligands to bind it tightly; regardless which type of matrix was used in the experiment, several perspectives have to be considered when choosing a proper support matrix, such as chemical stability and chemical inertness.

There are numerous techniques to separate protein based on its natural property, such as, separate protein based on various mass by ultracentrifugation or based on charge and mass ratio by SDS-polyacrylamide Gel electrophoresis (SDS-PAGE). Those purification methods would disclose some information about target protein; however, the result is less accurate and little vague to cause false positive. Compare to other purification method, the AC had various unique advantages, for instance, it recognized and purified biomolecule based on its biological function or chemical structure, and purification process took less time-consuming and accomplished with high recovery rate of target

The result of electrophoresis displayed target protein purified by two-step affinity chromatography and sialidase activity of the obtained protein fraction. (A), SDS-PAGE result, lane 1 represents the protein precipitated with 50% ammonium sulfate, lane 2 indicates the protein was eluted from mucin-sepharose column, lane 3 demonstrates the protein was unbound within mucin-sepharose column, lane 4 displays protein eluted from protein G-sepharose column. Lane 5 shows protein was unbound with G-sepharose column. Meanwhile, H and L indicated the heavy and light chain for IgGs respectively. (B) The result for western blotting, it shows IgGs was successful collected and reacted with anti-human IgG, lane 2 result. (C) The sialidase activity of the obtained protein fraction. IgGs was eluted from column and had relative high sialidase activity result, lane 2 and lane 4 respectively.7