Detection of an Antigen on an Immunoblot

Detection of the antigen on an immunoblot can be achieved by either enzyme-based detection systems or using detection reagents labeled with fluorochromes. Two major types of enzyme-based detection systems are used in immunoblotting based on either horseradish peroxidase (HRP)-or alkaline phosphatase (AP)-coupled antibodies, and a range of soluble substrates that yield insoluble colored products (chromogenic detection) or generate light (chemiluminescent detection). This protocol describes chromogenic detection with AP as well as both chromogenic and chemiluminescent methods of detection with HRP. Before the detection step, the nitrocellulose or polyvinylidene fluoride (PVDF) membrane is incubated with the antigen using enzyme-conjugated primary antibodies or secondary detection reagents (such as species-specific anti-immunoglobulin antibodies, streptavidin, or Protein A) and washed. An appropriate substrate solution is applied to the membrane, and the signal is detected in the course of the enzymatic reaction, resulting either in development of insoluble colored products (chromogenic substrates) or in generation of light (chemiluminescent substrates). This protocol also describes use of detection reagents labeled with fluorochromes. The nitrocellulose or PVDF membrane incubated with fluorochrome-labeled detection reagents is rinsed with PBS and processed for image capturing with appropriate imaging equipment.