In this procedure, a large well is used to separate the sample by PAGE and lanes are created on the membrane containing immobilized protein with the use of a manifold [134] . Compatible combinations of primary antibodies are predetermined, with the criterion of being able to identify proteins that do not comigrate. Different combinations of primary antibodies are added to each well, with appropriate dilutions of each primary antibody so that expressed proteins are detected in a single condition. The scalability of the system depends on defining suitable combinations of primary antibodies, with up to 1000 antibodies in 200 lanes being used in the largest screens. Detection software is used to identify proteins based on their expected and observed gel mobility. Unlike 2D PAGE and HPLC-MS/MS, large-scale western blotting only identifies proteins for which antibodies are already available. While this is not an appropriate screen for identifying uncharacterized proteins, it greatly simplifies the verification and functional analyses of proteins that are detected. In addition, this approach is highly flexible, and can be focused to particular sets of proteins or protein function, such as cell signaling molecules. Importantly, the foundation of this approach is the large amount of data on individual antibodies, which are already available and characterized in the literature [135] .