What are the methods used for validating antibodies in western blotting?

The methods for antibody validation in western blotting, include: genetic controls, testing across multiple cell lines, proteomic approaches, and independent-epitope methods. Among these, knock-out (KO) validation is considered the preferred method. The KO method involves using cells or organisms where the gene encoding the target protein has been knocked out or genetically modified to prevent its expression. Additionally, antibodies can be tested against lysates from various cell lines expressing different levels of the target protein. Mass spectrometry-based proteomic analysis can be used to confirm the identity of the target protein recognized by the antibody. Independent-epitope methods involve using antibodies raised against different epitopes of the target protein or antibodies from different sources targeting the same epitope. It is important to note that a combination of approaches should be used to ensure the reliability of an antibody for specific assays.