Detection of protein phosphorylation and charge isoforms using vertical one-dimensional isoelectric focusing gels

Abstract

During many biological responses, changes in protein modifi cations (e.g., phosphorylation) are often more critical than changes in protein abundance in determining the outcome of cellular responses. These important regulatory changes can alter a protein’s location, activity, or binding partners. Monitoring modifi cations such as phosphorylation is often impeded, or even prevented, because of the need for specialized reagents and equipment that are expensive and/or time-consuming to produce. However, many protein modifi cations alter the isoelectric point (p I) of a protein. Therefore, we developed a denaturing, onedimensional isoelectric focusing (IEF) procedure that separates proteins based on their p I to resolve different isoforms, allowing a relatively simple strategy for detecting changes in protein modifi cations. Although similar results can be achieved by two-dimensional gel electrophoresis, the method described here uses a multi-well SDS-PAGE format that allows many more samples to be assayed within a single gel, thereby greatly decreasing both the time and cost needed to assess modifi cations of a single protein in response many different treatment conditions. To increase the sensitivity of detection, we also optimized a procedure to transfer proteins from these gels to membranes for subsequent immunodetection. This combination of techniques provides the means of interrogating the number and stoichiometry of isoforms from total protein extracts without a priori knowledge of which modifi cation may occur.