2D Gel Electrophoresis to Address Biological Issues

Abstract

Two-dimensional (2D) gel electrophoresis is a high-resolution technique for the study of proteome. This chapter describes how it can be applied to characterize specific differences in the proteome profile of breast cancer cells following gene target interference. The proteome is the complete set of proteins encoded by a genome, and proteomic analysis consists in profiling the whole proteins expressed in a given cell, tissue, organ, or organism. Proteomic expression has the main purpose of qualitatively and quantitatively comparing proteins expressed under physiological and/or pathological conditions. Although it is not the unique approach used in modern proteomics, two-dimensional electrophoresis (2DE) is unrivaled allowing simultaneous separation of thousands of proteins and the detection of posttranslational modification, not predictable through genome analysis. 2DE combines two physical principles to separate complex protein mixtures: the isoelectric point and the molecular weight. The result is a gel map in which each protein isoform present in the sample can be visualized as a spot, analyzed, quantified, and identified by mass spectrometry analysis. Here we outline features and advantages of the 2DE-based proteomic approach and we describe how 2DE meets biochemistry and molecular biology to address specific issues.

Daytime Restricted Feeding Affects Day–Night Variations in Mouse Cerebellar Proteome

“…Post-translational modifications of proteins generally alter protein activity levels and/or half-lives. Because phosphorylation and acetylation trigger changes in protein isoelectric point, protein variants due to such modifications can be segregated from native proteins in different protein spots using twodimensional electrophoresis (Scumaci and Cuda, 2019). Here, G6pdx has been identified in two different protein spots, one affected by time-of-day only (see above), and another one affected by feeding conditions only (see below).…”