Affinity Chromatography-I

Note : Affinity chromatography will be covered in two lectures. The first lecture will cover general discussion while second will be more specific.

(1.) Introduction
(2.) General Principle of Affinity Chromatography
(3.) Procedure
(4.) Specific examples of Affinity Purification
(5.) Application of Affinity Chromatography in Proteomics

1. Introduction

Affinity chromatography was introduced almost 50 years back as a powerful tool for purification of biologically active molecules like proteins. This technique has revolutionary impact on modern biological sciences such as molecular biology, biochemistry, medicine and biotechnology. This technique exploits molecular recognition principle of a biological compound to be separated by the specific ligand to purify it from a mixture of compounds.

The affinity chromatography is a type of liquid chromatography for the separation and specific analysis of sample components. This type of chromatography makes use of a reversible "biological interaction" (molecular recognition) for the separation and analysis of specific analytes within a sample e.g. enzyme with an inhibitor and antigen with an antibody. One of the components, the ligand is immobilized onto a solid matrix, which is then used to selectively purify the target protein. Including a competing ligand in mobile phase or changing pH that elutes the target protein out. For example, Ni-Affinity chromatography is applied for the purification of 6xHis tagged proteins in which Ni is the chelating metal which is attached on NTA matrix (more detail of Ni-Affinity chromatography in next lecture).