b'Western blotting Transfer and detectionAfter electrophoresis, the separatedHorseradish peroxidase (HRP) or alkaline phosphatase (AP) proteins are transferred or blotted onto aare the most popular enzymes conjugated to antibodies used in the western blot workflow. After incubating the second matrix, generally a nitrocellulose ormembrane with the detection antibody or antibodies, if an polyvinylidene difluoride (PVDF) membrane.enzyme-conjugated antibody was utilized, an appropriate substrate (chromogenic or chemiluminescent) is added Next, the membrane is blocked to minimizeand that results in a detectable product. A popular potential nonspecific binding of antibodies tosubstrate of choice is a chemiluminescent substrate that, when combined with the enzyme, produces light as a the surface of the membrane.by-product. With the chemiluminescent substrate, the light output can be captured on X-ray film or an imaging instrument. In recent years, fluorescent detection became Detailed procedures vary widely for the detection steps ofa popular alternative to enzymatic detection since it allows the western blot workflow. One common variation involvesfor more quantitative data analysis. Fluorescent detection direct vs. indirect detection methods. In both the directutilizes dye-labeled primary antibodies or dye-labeled and indirect detection methods, the blocked membrane issecondary antibodies, and the signal output is captured probed with an antibody (primary antibody) specific to theon an appropriate imaging system. Regardless of the protein of interest (antigen). In direct detection techniques,detection system used, the intensity of the signal should this primary antibody is enzyme conjugated or labeled withcorrelate with the abundance of the antigen on the blotting a fluorophore. However, in indirect detection techniques,membrane.the blocked membrane is probed first with an antibody (primary antibody) that is specific to the antigen followedWe offer a wide range of reagents, kits, equipment, and by another antibody (secondary antibody) raised againstantibodies to facilitate every step of western blot analysis. the host species of the primary antibody. This secondary antibody is often enzyme conjugated or labeled with a fluorophore. The direct method is not widely used, as most researchers prefer the indirect detection method for a variety of reasons, including workflow flexibility, broader choice of conjugates, and the potential for cost savings and increased sensitivity.Techniques and tools for publication-quality resultsDownload at thermofisher.com/westernhandbook72'