MBB 222 Study Guide - Final Guide: Primary And Secondary Antibodies, Immunoassay, Western Blot

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Document Summary

This is an immunoassay technique that can be used for protein detection and quantification. Protein mixture is run on an sds-page. Protein bands are transferred to a polymer membrane using an electric field (electroblotting) Primary antibody is added, followed by secondary that is enzyme attached (will react with substrate to produce a coloured product, as in elisa) Blot is developed by adding substrate, the protein bands that contain the antigen will appear on the blot. Primary antibody is specific to and binds to the antigen. Secondary antibody is specific to and binds to the primary antibody. Is enzyme attached, so that a certain substrate can be added allowing for a coloured product to be formed. The coloured product formed by substrate added to a solution with secondary antibodies, showing the presence of primary antibodies bound to antigens. Protein (antigen) of interest is immobilized in a well and an primary antibody that is specific to this antigen is applied.