In a Western blot procedure, what is added after transferring proteins to the membrane?

In a Western blot procedure, after transferring proteins to the membrane, secondary antibodies are added to specifically bind to the primary antibodies that were previously applied to the membrane. The primary antibodies are used to recognize and attach to the target proteins of interest. The addition of secondary antibodies is crucial for the detection process because these antibodies are typically conjugated with an enzyme or a fluorophore. This conjugation allows for the visualization of the proteins that have been bound by the primary antibodies, often through chemiluminescent or fluorescent methods. This step amplifies the signal and makes it possible to quantitatively assess the presence of specific proteins in the sample, which is essential for analyzing protein expression levels.

Detergents, sample buffer, and cofactors, while important in different stages of the overall protein analysis process, do not follow directly after the transfer step in the Western blotting technique. Detergents are usually part of the sample preparation and lysis steps, sample buffer is used before electrophoresis, and cofactors might be relevant during enzymatic assays but are not directly related to the Western blot procedure itself.