Optimized Protocol to Make Phospho-Specific Antibodies that Work

Abstract

Phosphoproteins are considered to be among the most important proteins in the body. They are the proteins that regulate almost all cell processes from cell division in cancer to neuronal signal transduction in learning and memory. This review will describe the development of a revolutionary immunochemical technique that produces antibodies that bind to target proteins only when the protein is in the phosphorylated state. These phospho-specific antibodies can thus be used to track the activity of a protein, not simply its level of expression. In this review, we will discuss both the design of the phosphopeptide immunogen and immunization. The affinity purification of the phospho-specific antibody as well as the methods most suitable for characterizing the phosphospecificity of the antibody will be described here. Taken together, these methods will cover the key procedures and protocols required to produce a phospho-specific antibody that works.