Biotechnology and Bioinformatics (1+1)

Principle

The basic principle be hind Western blotting and immune detection is that of antigen and antibody interaction. An antigen bound to the primary anti body is detected by a secondary antibody labeled with an enzyme, when the substrate is added gives a colored insoluble product, w hich can be detected visually.

The proteins from bacterial cells[hemolysin], tissue culture or any other source are first separated by SDS -PAGE and are transferred or blotted from the gel to anitro cellulose or nylon membrane that binds proteins non specifically. When this membrane is treated with primary antibody directed against desired protein antigen, if the specific antigens are present , then the anti body will bind to them . T hen th e added Enzyme -labeled secondary antibody recognizes and binds to the primary antibody . The antigen-primary antibody ­ secondary antibody-enzyme complex is detected when the enzyme converts a soluble, color less substrate into an in soluble, colored product . Thus the colored bands appear on the white membrane wherever a protein antigen interacts with the primary anti body