Blocking non-specific binding sites on the membrane

Antigen-primaiy monoclonal antibody complexes are recognized by a second-aiy anti-immunoglobulin antibody conjugated to a marker to provide a means of detection. Enhanced sensitivity is achieved in this signal amplification step as each primary monoclonal antibody is recognized by many molecules of the secondajy antibody. 

Secondary antibodies labelled with radioisotopes (typically I) are detected by autoradiography, and the signals are quantitated by densitometric scanning of the film. Alternatively, radiolabelled secondary antibodies result in excitation of a Phosphor storage screen, and the signals are detected and quantitated by Phosphorimaging. 

Secondary antibodies conjugated to horseradish peroxidase (HRP) or alkaline phosphatase (AP) are detected in an enzymatic reaction  

After transfer or staining, submerge the membrane blot in blocking buffer in a container. 

Incubate the membrane in blocking buffer for at least 2 h at room temperature or overnight at 4 °C with constant agitation on a shaker. 

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