Protein mapping by combined gel electrofocusing and electrophoresis: application to the study of genotypic variations in wheat gliadins.
01 Aug 1970-Biochemical Genetics (Kluwer Academic Publishers-Plenum Publishers)-Vol. 4, Iss: 4, pp 509-516
TL;DR: The technique revealed considerable differences in gliadin composition between different varieties of bread wheats, and showed that the composition is affected qualitatively by the removal of the D-genome for the hexaploid wheats Prelude, Rescue, and Thatcher.
Abstract: Gliadin was fractionated into a two dimensional map of over 40 components by gel electrofocusing in the first dimension, followed by starch gel electrophoresis in the second dimension. Many apparently single zones, fractionated by either procedure alone, were shown to consist of several components. The technique revealed considerable differences in gliadin composition between different varieties of bread wheats, and showed that the composition is affected qualitatively by the removal of the D-genome for the hexaploid wheats Prelude, Rescue, and Thatcher, and by the removal of the short arm of chromosome 1B of Chinese Spring.
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TL;DR: The fact that a high number of protein spots can be evaluated by a single and comparatively simple experiment suggests that this method may be useful as an assay system for induced point mutations.
Abstract: The protein-mapping method which combines isoelectric focusing in acrylamide gel and gel electrophoresis was previously used mainly for the separation of plant proteins and human serum proteins. We investigated with this technique soluble proteins of mouse tissues (whole embryos, the liver of fetal and adult mice, kidneys) and the proteins of mouse serum. The technique was tested under a number of different conditions to find those best for our purpose; they may represent some general improvements in the method. The protein patterns show high resolution and excellent reproducibility. About 275 spots were found for fetal liver, about 230 for whole embryos (day 14 p.c.) and about 100 for serum. The fact that a high number of protein spots can be evaluated by a single and comparatively simple experiment suggests that this method may be useful as an assay system for induced point mutations. The protein patterns demonstrated are compared and discgs of dominant lethal examinations after acute and subacute application of these three substances.
1,166 citations
Cites background from "Protein mapping by combined gel ele..."
...The two-dimensional separation of proteins by combination of isoelectric focusing and slab gel electrophoresis was developed in 1968--69 (Margolis and Kenrick, cited by Wrigley, 1968; Dale and Latner, 1969; Macko and Stegemann, 1969) and was mainly used for the investigation of certain plant proteins (Macko and Stegemann, 1969; Wrigley, 1970; Wrigley and Shepherd, 1973; Stegemann et al., 1973) and human serum proteins (Dale and Latner, 1969; Kenrick and Margolis, 1970; Domschke et al....
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TL;DR: It is shown how effectively 2‐DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross established by the European Backcross Collaborative Group for mapping the mouse genome.
Abstract: The two-dimensional electrophoresis (2-DE) technique developed by Klose in 1975 (Humangenetik 1975, 26, 211-234), independently of the technique developed by O'Farrell (J. Biol. Chem. 1975, 250, 4007-4021), has been revised in our laboratory and an updated protocol is presented. This protocol is the result of our experience in using this method since its introduction. Many modifications and suggestions found in the literature were also tested and then integrated into our original method if advantageous. Gel and buffer composition, size of gels, use of stacking gels or not, necessity of isoelectric focusing (IEF) gel incubation, freezing of IEF gels or immediate use, carrier ampholytes versus Immobilines, regulation of electric current, conditions for staining and drying the gels - these and other problems were the subject of our concern. Among the technical details and special equipment which constitute our 2-DE method presented here, a few features are of particular significance: (i) sample loading onto the acid side of the IEF gel with the result that both acidic and basic proteins are well resolved in the same gel; (ii) use of large (46 x 30 cm) gels to achieve high resolution, but without the need of unusually large, flat gel equipment; (iii) preparation of ready-made gel solutions which can be stored frozen, a prerequisite, among others, for high reproducibility. Using the 2-DE method described we demonstrate that protein patterns revealing more than 10 000 polypeptide spots can be obtained from mouse tissues. This is by far the highest resolution so far reported in the literature for 2-DE of complex protein mixtures. The 2-DE patterns were of high quality with regard to spot shape and background. The reproducibility of the protein patterns is demonstrated and shown to be thoroughly satisfactory. An example is given to show how effectively 2-DE of high resolution and reproducibility can be used to study the genetic variability of proteins in an interspecific mouse backcross (Mus musculus x Mus spretus) established by the European Backcross Collaborative Group for mapping the mouse genome. We outline our opinion that the structural analysis of the human genome, currently pursued most intensively on a worldwide scale, should be accompanied by a functional analysis of the genome that starts from the proteins of the organism.
716 citations
TL;DR: An important parameter, the isoelectric point (pI), is defined, which gives information not only about the composition and conformation of macromolecules but also allows a rational approach to further experimental manipulations.
371 citations
TL;DR: In this paper, the authors present three stabilizing techniques, density gradient, polyacrylamide-gel, and zone convection electrofocusing, to counteract convective remixing of the separating components.
Abstract: Publisher Summary The method of isoelectric focusing is making new inroads in the study of proteins from various sources. The method requires a stable pH gradient between the anode and the cathode in an electrolysis cell. This is obtained by the electrolysis of a water solution of a mixture of suitable low molecular weight ampholytes, called carrier ampholytes. When proteins are put into such a system each protein migrates to, and focuses at, its isoelectric point, p I , independent of where it was put into the apparatus at the start. Therefore, the volume of the protein sample solution to be applied is not critical and even dilute protein solutions of comparatively large volumes can be applied. Isoelectric focusing requires some means to counteract convective remixing of the separating components. At present three stabilizing techniques are used—(1) density gradient, (2) polyacrylamide-gel, (3) zone convection electrofocusing. The first technique is the most common, and an apparatus for 110 ml and another for 440 ml are commercially available. The second is of rapidly growing importance and is especially valuable for analytical separations and comparative work. The third technique is very simple, as it is in principle performed in a serpentine channel and does not require such additives as sucrose or gel.
292 citations
TL;DR: An apparatus and method are described for polyacrylamide gel electrophoresis of gliadins since the gliadin pattern (electrophoregram) is a genotypic character, and offers a promising means for identifying wheat cultivars.
Abstract: An apparatus and method are described for polyacrylamide gel electrophoresis of gliadins. Since the gliadin pattern (electrophoregram) is a genotypic character, electrophoresis offers a promising m...
289 citations
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1,224 citations
"Protein mapping by combined gel ele..." refers methods in this paper
...(Vesterberg and Svensson, 1966) in combinat ion with a procedure, such as cellulose...
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TL;DR: The method has been applied to myelomatosis and cirrhosis and appears capable of separating IgA from IgG and can be applied to other biological fluids including urine and cerebrospinal fluid.
101 citations
94 citations
"Protein mapping by combined gel ele..." refers methods in this paper
...Combined gel electrofocusing and electrophoresis has already been used to fractionate and characterize serum proteins (Wrigley, 1968; Dale and Latner, 1969; Kenrick and Margolis, 1970) and potato proteins (Macko and Stegeman, 1969)....
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89 citations
"Protein mapping by combined gel ele..." refers methods in this paper
...Because of the resolution obtainable with gel electrophoresis, the procedure has been used extensively for examining variations in gliadin composition of different wheat genotypes in relation to breadmaking quality, taxonomy, evolution, and genetic constitution (Johnson et al., 1967; Doekes, 1968; Boyd et al., 1969; Dronzek et al., 1970)....
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TL;DR: The work reported here was designed to test as rigorously as possible whether the second kind of change occurs within the gliadin class of proteins within a variety.
Abstract: CHEMICAL and physical investigations have shown that gluten composition differs from one wheat variety to another1–5. The total protein as a percentage of the weight of grain of a particular variety is affected by environmental factors and may be twice as great in some samples as in others grown in different conditions. Variations of this kind within a variety can result in an alteration in the proportions of the broad protein classes6,7 and could alter the distribution of components within classes. The work reported here was designed to test as rigorously as possible whether the second kind of change occurs within the gliadin class of proteins. It is desirable to know whether or not this type of change can occur if information on the composition of the gliadin is to be readily applicable to genetic investigations of inheritance of wheat quality.
44 citations
"Protein mapping by combined gel ele..." refers background in this paper
...It has been shown previously that gliadin composition, determined by starch gel electrophoresis or column chromatography, is independent of growth conditions or grain protein content, but is controlled solely by genetic constitution (Lee and Ronalds, 1967)....
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