Procedure of Affinity chromatography

The target molecule in a sample-specific for a particular ligand determinant can be isolated by the use of affinity chromatography. The separation of the target molecule the affinity separation occurs in four steps.

Following is the four-step of the affinity chromatography:

Here the four steps explain by separation of antibodies from the serum. The antibodies in a serum sample specific for a particular antigenic determinant can be isolated by the use of affinity chromatography.

Procedure of Affinity chromatography

Step:1

—  An immunoadsorbent is prepared. This consists of a solid matrix to which the antigen (shown in blue) has been coupled (usually covalently). Agarose, sephadex, derivatives of cellulose or other polymers can be used as the matrix.

Step:2

—  The serum is passed over the immunoadsorbent. As long as the capacity of the column is not exceeded, those antibodies in the mixture specific for the antigen (shown in red) will bind and be retained. Antibodies of other specificities (green) and other serum proteins (yellow) will pass through unimpeded.

Step:3

—  Elution. A reagent is passed into the column to release the antibodies from the immunoadsorbent. Buffers containing a high concentration of salts and/or low pH are often used to disrupt the noncovalent interactions between antibodies and antigen. A denaturing agent, such as 8 M urea, will also break the interaction by altering the configuration of the antigen-binding site of the antibody molecule.

—  Another approach is to elute with a soluble form of the antigen. These compete with the immunosorbent for the antigen-binding sites of the antibodies and release the antibodies to the fluid phase.

Step:4

—  The eluate is then dialyzed against, for example, buffered saline in order to remove the reagent used for elution.

Related Post:

Ligands in Affinity Chromatography

Procedure for Affinity Chromatography