Generation and utilization of phosphorylation state-specific antibodies to investigate signaling pathways.

Phosphorylation state-specific antibodies can be of great use, for example, in studying individual steps within a given signal transduction pathway. This unit presents a general approach to the generation and purification of phosphorylation state-specific antibodies. In addition to their ability to detect phosphorylation at a particular key site, these antibodies are often more sensitive for biochemical studies. Besides their application in immunoblotting procedures, activation state-specific antibodies can be used as immunohistochemical reagents. Thus, critical changes in phosphorylation can be monitored as described on an individual cell basis or in fixed tissue sections. Such antibodies can be used to address fundamental questions about signal transduction pathways during physiologic events that cannot be resolved by more conventional methodologies.