Affinity Chromatography

Affinity Chromatography is an essential method used to separate desired protein from complex mixtures, however it can also be used for studying P rotein- P rotein interaction. It is based on the affinity interaction among various groups of compatible proteins for its separation from complex mixtures. Affinity chromatography involves the retention of the desired sample within the column from the sample mixture applied . In this method one of the protein ( against which interaction is to be checked)  is coupled with suitable matrix in the chromatographic column.The sample mixture containing the protein of interest from suitable source is applied against it. Most of the proteins pass down through the column however, some proteins may get retained into it. The protein which comes into affinity bonding or interaction with the immobilized protein within the column gets retained and is further eluted by suitable treatment. The eluted sample is collected for further analysis (Fig. 1).

Care should be taken during the preparation of chromatographic column i.e .

• The coupling of protein with the matrix should not inhibit its functional activity otherwise it ...would become inactive and incapable of interacting and retaining the desired sample.
• Pure protein sample is to be used for column preparation in order to prevent unwanted protein  ...interaction.
• The amount of sample protein applied must not be too low to be unnoticeable  for detection ...within column and also must not be too high for many small proteins to get eluted without ...binding.