High Speed Isoelectric Focusing of Proteins Enabling Rapid Two-Dimensional Gel Electrophoresis (original) (raw)
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Two-dimensional electrophoresis of proteins in an immobilized pH 4-12 gradient
Electrophoresis, 1998
For checking theoretical two-dimensional (2-D) maps derived from sequenced genomes, indicating that nonnegligible amounts of proteins up to pH 12 are to be expected, a wide-range immobilized pH 4-12 gradient was generated. Depending on the extraction method of sample preparation, proteins with pls up to pH 12 are detected in a single gel. Highly reproducible protein patterns focused to the steady state with round-shaped spots up to pH 12 are obtained with the standard protocol originally described in 1988 (Gorg et al., Electrophoresis 1988, 9, 531-546).
ELECTROPHORESIS, 2005
We previously demonstrated the separation of proteins by isoelectric focusing (IEF) over pH 4-8 immobilized pH gradients (IPGs) over 54 cm (Poland et al., Electrophoresis 2003, 24, 1271. Here we show that similar results can be conveniently achieved using commercially available IPGs of appropriate pH ranges positioned end-on-end in series during electrophoresis, which we term 'daisy chain IEF'. Proteins efficiently electrophorese from one IPG to another during IEF by traversing buffer-filled porous bridges between the serial IPGs. A variety of materials can function as bridges, including paper, polyacrylamide gels or even IPGs. The quality of two-dimensional (2-D) protein patterns is not apparently worse than that generated by conventional IEF using the same individual IPGs. A major advantage of this method is that sample is consumed efficiently, without the requirement for preliminary steps, such as chamber IEF. This advantage is pronounced when working with extremely limited sources of samples, such as with clinical biopsies or cellular subfractions. The present study was limited by the commercial availability of suitable pH gradients. Proteomics analyses could be further improved if commercial vendors would manufacture IPGs with suitable pH ranges to achieve high resolution (,100 cm) IEF separation of proteins in one electrophoretic step over the pH range 2-12. Electrophoresis 2005, 26, 3185-3190 3185
Two-dimensional electrophoresis of membrane proteins
Analytical and Bioanalytical Chemistry, 2007
resolving technique for arraying proteins by isoelectric point and molecular mass. To date, the resolving ability of 2-DE for protein separation is unsurpassed, thus ensuring its use as the fundamental separation method for proteomics. When immobilized pH gradients (IPGs) are used for isoelectric focusing in the first dimension, excellent reproducibility and high protein load capacity can be achieved. While this has been beneficial for separations of soluble and mildly hydrophobic proteins, separations of membrane proteins and other hydrophobic proteins with IPGs have often been poor. Stimulated by the growing interest in proteomics, recent developments in 2-DE methodology have been aimed at rectifying this situation. Improvements have been made in the area of protein solubilization and sample fractionation, leading to a revamp of traditional approaches for 2-DE of membrane proteins. This review explores these developments.
Electrophoresis, 1997
Membrane proteins were separated by high resolution two-dimensional (2-D) electrophoresis. On isoelectric focusing (IEF) with immobilized pH gradients severe protein losses in the resulting 2-D map were observed when compared with carrier ampholyte-based IEF. This has been noticed for two different biological systems, namely the chloroplast envelope of spinach and the endocytic vesicles from Dictyostelium discoideum. The possible mechanisms of these losses on immobilized pH gradients are discussed?
The current state of two-dimensional electrophoresis with immobilized pH gradients
Electrophoresis, 2000
The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Görg et al., Electrophoresis 1988, 9, 531±546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or ingel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.
Large-scale electrophoresis for protein purification: exploiting isoelectricity
1988
Preparative electrophoresis methods (including isoelectric focusing in immobilized pH gradients) in gel phases are characterized by low loadings (barely a few mg protein per ml matrix), low recoveries (rarely exceeding 70%), and heavy contamination from neurotoxic gel materials (the unreacted gel monomers and ungrafled oligomers). These drawbacks can be minimized by a version of isoelectric focusing in which the need for protein of interest to pass the gel is eliminated: only the contaminants traverse the gel. This is achieved by circulating a liquid sample between two gels held at controlled pHs. The method can provide: (1) high rate of sample processing (up to 1 g h-l); (2) high purification (in general to charge homogeneity); and (3) high recoveries (>95%). A large-scale membrane apparatus has been built, with a cross-sectional diameter of 9 cm. Large Pt electrode disks provide even current flow. In this electrolyser, 10 g of Eglin C (produced by recombinant DNA technology) have been purified to homogeneity in around 10 h from 1 1 of a partially enriched preparation.
Electrophoresis, 1985
Factors affecting the range of pH gradients in the isoelectric focusing dimension of two-dimensional gel electrophoresis: The effects of reservoir electrolytes Max-Planck-Institut fur molekulare Genetik, Berlin and loading procedures We have examined methods for broadening and stabilising pH gradients used in the first dimension of two-dimensional gel electrophoresis. The replacement of typical strong electrolytes with weak electrolytes as reservoir anolyte and catholyte allows the generation of broad (3.5 pH unit) gradients that are stable for at least 28 000 volt x hours (Vh). Protein patterns form within 10 000 Vh are stable throughout the subsequent focusing. Large quantities ofprotein, up to 6 mg, can be loaded onto such gels by mixing samples into the gel prior to polymerisation. We found no evidence for extensive modification of proteins because of exposure to the polymerisation reaction. The pH gradient formed in strong electrolytes in the typical O'Farrell gel system (J. Bid. Chem. 1975,250,4007-4021) could also be broadened ifthe samples were again mixed into the gel before polymerisation. This last effect appears to be due to an effect of the sample buffer used upon end loading of samples.