Azure BiosystemsChemiluminescence is a popular indirect detection method for Western blotting. This technique is very good at answering the question, “Is my protein there or not?” and can provide a qualitative answer to the question “Is the amount of my protein different between these two samples?”
In chemiluminescent detection, the primary antibody binds to the target protein on the membrane, and the location of the primary antibody is detected using a secondary antibody conjugated to an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (AP).
A substrate for the enzyme is added and when the enzyme acts on the substrate, light is emitted (Figure 1.6). The light can be detected using a CCD camera or x-ray film (Figure 1.7). The sensitivity of detection depends on the choice of substrate—commercially available substrates for HRP can detect proteins in the femtogram range.
Chemiluminescent detection is often used because it is specific, easy to perform, and highly sensitive. The labeled secondary antibody can be used across multiple experiments to detect any primary antibody of the correct species, making the approach cost-effective.
Flexible gel doc imaging system for Western blotting and DNA gels
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