The evolution of two-dimensional gel electrophoresis - from proteomics to emerging alternative applications.

Two-dimensional gel electrophoresis (2-DE) is a technique that has been widely applied in a variety of proteomics studies. It is capable of resolving complex protein mixtures into individual protein spots based on their isoelectric point and molecular weight, enabling large-scale analysis of protein expression patterns for deciphering their changes in different biological conditions. 2-DE is a powerful tool that empowers researchers to perform differential qualitative and quantitative proteome analysis and is particularly advantageous for characterizing protein isoforms and post-translationally modified proteins. Despite its popularity as the workhorse for proteomics in the past few decades, it has been gradually displaced by the more sophisticated and high-performance mass spectrometry-based methods. However, there are several variations of the 2-DE technique that have emerged as promising approaches that shine new light on specific niches that 2-DE could still contribute. In this review, we first provide an overview of the applications of 2-DE, its merits and pitfalls in the current proteomic research arena, followed by a discussion on several alternative approaches for potential future applications.

Challenges, current status and future perspectives of proteomics in improving understanding, diagnosis and treatment of vascular disease.

Technical advances have seen the rapid adoption of genomics and multiplex genetic polymorphism identification to research on vascular diseases. The utilization of proteomics for the study of vascular diseases has been limited by comparison. In this review we outline currently available proteomics techniques, the challenges to using these approaches and modifications which may improve the utilization of proteomics in the study of vascular diseases.

Features and applications of blue-native and clear-native electrophoresis.

1-D native electrophoresis is used for the separation of individual proteins, protein complexes, and supercomplexes. Stable and labile protein-protein interactions can be identified depending on detergent and buffer conditions. 1-D native gels are immediately applicable for in-gel detection of fluorescent-labeled proteins and for in-gel catalytic activity assays. 1-D native gels and blots are used to determine native mass and oligomeric state of membrane proteins. Protein extracts from 1-D native gels are used for generation of antibodies, for proteomic work, and for advanced structural investigations. 2-D separation of subunits of protein complexes by SDS-PAGE is mostly used for immunological and proteomic studies. Following the discussion of these general features, specific applications of native electrophoresis techniques in various research fields are highlighted: immunological and receptor studies, biogenesis and assembly of membrane protein complexes, protein import into organelles, dynamics of proteasomes, proteome and subproteome investigations, the identification and quantification of mitochondrial alterations in apoptosis, carcinogenesis, and neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, and the vast variety of mitochondrial encephalomyopathies.

Improvement deoxyribo nucleic acid spots classification in polyacrylamide [correction of polyacrilamide] gel images using photometric normalization algorithms.

A comparative study of four enhancement algorithms traditionally used in computer vision for photometric normalization of images affected by illumination changes is presented in this paper. We experimented with the performance of these approaches to reduce the low frequency multiplicative noise that is produced as a result of a non-homogeneity illumination or a non-homogeneity developed chemical process in polyacrylamide gel electrophoresis images for the purpose of automatic classification of deoxyribo nucleic acid (DNA). The algorithms are tested on a database and their results are compared in a system for feature extraction and DNA classification.

A primary culture system for biochemical analyses of neuronal proteins.

Low-density cultures of embryonic rat hippocampal neurons have been widely used to investigate localization and function of neuronal proteins using immunocytochemistry and electrophysiology. These cultures provide a relatively homogeneous population of hippocampal pyramidal neurons and interneurons compared to post-natal mixed neuron/glial cultures from hippocampus, cerebral cortex, and cerebellum. However, the limited quantity of neurons and the difficulty in harvesting adequate amounts makes biochemical analyses of endogenous neuronal proteins in these low-density cultured neurons difficult. Here, we provide detailed methods to prepare cultures of embryonic rat hippocampal neurons suitable for biochemical analyses of both endogenously and exogenously expressed proteins. The procedures described here are also suitable for comprehensive studies of expression, localization, post-translational modification, and function of neuronal proteins in the same neuronal culture system.