Abstract

BackgroundTwo-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae.ResultsProtein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI > 7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load.ConclusionsComparison of broad range (pH 3–10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH 4–7) IPG technique is a method of choice for the analysis of acidic proteins.

Highlights

  • Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics

  • Our results suggest that the broad range pH 3–10 IPGbased 2DE method suffers from the same limitations as cathodic isoelectric focusing (CIF) technique of the non-equilibrium pH gradient electrophoresis (NEPHGE) method

  • First dimension immobilized pH gradient (IPG) (Invitrogen) and NEPHGE (WITAvision) techniques were directly compared in twodimensional gel electrophoresis experiment using the same format mini-gels and the same samples of yeast whole cell lysates

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Summary

Introduction

Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. There are two different first dimension separation techniques: the method of Klose [3] and O’Farrell [4], where the pH gradient is formed via carrier ampholytes (CA) (amphoteric, oligoaminooligocarbonic acids with high buffer capacity at their pI) during the focusing process and the method described by Bjellqvist and Görg [5,6,7] using immobilized pH gradient (IPG). Due to its simple handling and commercialization, IPG-based IEF is typically used for 2DE-based proteome analysis and has widespread applications. CA-based IEF, being a labour-intensive technique, failed to achieve widespread application, but is still used in more specialized laboratories

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