Showing papers in "Electrophoresis in 1988"
TL;DR: An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G‐250 and R‐250 to combine the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining.
Abstract: An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
2,638 citations
726 citations
TL;DR: A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit, which results in a considerable increase in sensitivity without the need for a recycling step.
Abstract: A new modification of silver staining of proteins in sodium dodecyl sulfate polyacrylamide gels is adapted to automated staining in PhastSystem Development Unit. The use of a reduction step, after fixation, with thiosulfate in alcoholic sodium acetate buffer results in a considerable increase in sensitivity without the need for a recycling step. The detection limit is tenfold lower than in the silver staining procedure recommended so far for PhastSystem and corresponds to 0.05–0.1 ng protein per band. Total staining time with the new procedure is 75 min.
696 citations
TL;DR: Modifications of the procedure for silver staining of proteins in polyacrylamide gels have been improved by sensitizing the gels with sodium dithionite instead of sodium thiosulfate and by equilibration in water after fixation and prior to sensitization.
Abstract: High sensitivity and low background, the attractive characteristics of the procedure of Blum et al., Electrophoresis 1987, 8, 93-99, for silver staining of proteins in polyacrylamide gels have been improved by sensitizing the gels with sodium dithionite instead of sodium thiosulfate and by equilibration in water after fixation and prior to sensitization. These modifications decrease the background and allow for a longer development period, which in turn increases sensitivity and color contrast. In addition, the colors of the spots are shifted toward colder tones when compared with the original method.
293 citations
TL;DR: Three different extraction procedures for two‐dimensional electrophoresis of plant proteins are compared and a trichloroacetic acid‐acetone procedure allowing the direct precipitation of total proteins is compared.
Abstract: Three different extraction procedures for two-dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Tris-buffer, (ii) denaturing extraction in presence of sodium dodecyl sulfate at elevated temperature allowing the solubilization of membrane proteins in addition to a recovery of soluble proteins, and (iii) a trichloroacetic acid-acetone procedure allowing the direct precipitation of total proteins.
197 citations
TL;DR: The three different simple repetitive oligonucleotide probes were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases and revealed informative fingerprints that can be used for individual identification, e.
Abstract: The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 × 10−8. The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e. g. in paternity testing and in forensic applications. In most of these experiments 32P-labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P-labelled probe (GACA)4.
147 citations
TL;DR: Two‐dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures.
Abstract: Two-dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time-consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low microgram range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two-dimensional electrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R-250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N-terminal sequence determination.
123 citations
TL;DR: Highly reproducible two‐dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.
Abstract: Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.
122 citations
TL;DR: Examples of the differences between the microheterogeneity patterns of transferrin in several biological fluids and the changes that can be observed in diseases such as rheumatoid arthritis, idiopathic hemochromatosis and Kahler's disease are presented.
Abstract: The heterogeneity of human transferrin results from (i) differences in iron content, (ii) genetic polymorphism and (iii) differences in the carbohydrate moiety. This article primarily deals with the last phenomenon, the microheterogeneity of human transferrin. Owing to the comparatively simple carbohydrate structure of human transferrin and the high resolving power of isoelectric focusing in immobilized pH gradients, microheterogeneous forms of transferrin can be separated. Differences between samples can be quantitated by crossed immunoelectrophoresis. Examples of the differences between the microheterogeneity patterns of transferrin in several biological fluids and the changes that can be observed in diseases such as rheumatoid arthritis, idiopathic hemochromatosis and Kahler's disease are presented. Special attention has been focused on changes occurring during pregnancy.
119 citations
TL;DR: Using phenol extraction from tobacco callus, extracts with a high protein content are prepared and it is demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.
Abstract: Using phenol extraction from tobacco callus, we have prepared extracts with a high protein content. These proteins were separated in cylindrical non-equilibrium pH gradient gels and visualized by dipping in sodium dodecyl sulfate (SDS)-containing solution. Three gel sections, each containing proteins previously detected as abundantly synthesized in tobacco mesophyll protoplasts and whose synthesis is reduced by auxin application, were excised from each gel and collected. These proteins were further separated on slab SDS gels and protein bands were excised after Coomassie Brilliant Blue R-250 staining and used to inject three rabbits. After one booster, highly specific antibodies were detected in their sera by ELISA and immunoblotting. Using these sera we have confirmed that the corresponding proteins are identical in callus and mesophyll protoplast and demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.
102 citations
TL;DR: The use of IPG gels focused to equilibrium should not only improve inter‐gel reproducibility and resolution but also the quality of the final 2‐D patterns with respect to background staining and horizontal streaking.
Abstract: Horizontal two-dimensional (2-D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P-40 (Gorg et al., Electrophoresis 1987, 8, 45-51), has been extended to analyze basic proteins of yeast cells focused under non-equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2-D patterns. Inclusion of 0.5% carrier ampholytes (CA) to the IPG gel (IPG-CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2-D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG-CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2-D gels using diluted IPG gels than with "standard" IPG gels. Optimization of 2-D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter-gel reproducibility and resolution but also the quality of the final 2-D patterns with respect to background staining and horizontal streaking.
TL;DR: The classification method and the preliminary results obtained with liver biopsy electrophoretograms are described and heuristic clustering is also compared to other classification techniques.
Abstract: The interpretation of two-dimensional gel electrophoresis (2-DGE) profiles can be facilitated by artificial intelligence and machine learning programs. We have incorporated into our 2-DGE computer analysis system (termed MELANIE-Medical Electrophoresis Analysis Interactive Expert system) a program which automatically classifies 2-DGE patterns using heuristic clustering analysis. This program is a step toward machine learning. In this publication, we describe the classification method and the preliminary results obtained with liver biopsy electrophoretograms. Heuristic clustering is also compared to other classification techniques.
TL;DR: Results are reported using PhastSystem for purity checking and characterization of monoclonal antibodies after affinity chromatography and also for determining their digestion rate with papain.
Abstract: PhastSystem, an integrated system for horizontal electrophoresis and isoelectric focusing in small gels, including automated staining and destaining, is described. Buffers for electrophoresis are supplied to the gel from buffer strips made of agarose. The separation bed is cooled by Peltier elements. All conditions of significance to the results, both during separation and development, are controlled by a microprocessor. For separation, nine programs are available, each with 9 steps; for development, there are nine programs with 20 steps each. In this paper, we also report results using PhastSystem for purity checking and characterization of monoclonal antibodies after affinity chromatography and also for determining their digestion rate with papain.
TL;DR: By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 μm thickness, which allowed the detection of small peptides with a detection limit of approximately 10 pg/section.
Abstract: A sensitive method is described for the detection of tissue peptides and proteins. They are separated by tissue isoelectric focusing using thin large-pore polyacrylamide gels, containing detergent and dimethylformamide, and are fixed with either glutaraldehyde or formaldehyde in gelatin-coated nitrocellulose membranes using press-blotting. The fixed peptide and protein antigens are visualized by immunoperoxidase staining. The spectrum of fixed tissue constituents may also be used to test antiserum reactivity and specificity in immunocytochemical staining procedures. Isoelectric focusing of 2 microL homogenates of the neurointermediate lobe of the pituitary allowed the immunodetection of peptides and proteins of various sizes and the determination of isoelectric points. However, direct application onto gels of small pieces of frozen tissue sections, sliced in a cryostat, appeared to be more efficient. By direct tissue isoelectric focusing of brain tissue, peptides were effectively eluted and separated from sections up to 100 microns thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.
TL;DR: Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining.
Abstract: Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.
TL;DR: Several high molecular weight polypeptides whose synthesis and presence in spinach leaf tissue were highly correlated with freezing tolerance were identified and suggested that they could play a role in cold tolerance mechanisms of spinach.
Abstract: Exposure of spinach (Spinacia oleracea) seedlings to 5 degrees C for several days has previously been shown to induce a greater tolerance to the stresses of extracellular freezing. Associated with this response to low temperature, termed cold acclimation, was a subtle shift in protein synthesis and altered polypeptide composition. Two-dimensional gel electrophoresis was used to study the changes in spinach leaf tissue protein synthesis in an effort to identify polypeptides that may play a central role in the induction of greater freezing tolerance. Through a combination of silver staining, in vivo labeling, and in vitro translation of mRNAs, we identified several high molecular weight polypeptides whose synthesis and presence in spinach leaf tissue were highly correlated with freezing tolerance. Synthesis of these polypeptides was elevated or induced during cold acclimation when freezing tolerance increases, but was rapidly reduced or halted during deacclimation when freezing tolerance declines. The close association of the synthesis of these polypeptides with the induction and loss of freezing tolerance suggested that they could play a role in cold tolerance mechanisms of spinach.
TL;DR: Different equilibration conditions of the first‐dimensional immobilized pH gradient gel strip prior to second‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis were evaluated and silver stained two‐dimensional patterns were obtained.
Abstract: Protocols for horizontal two-dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two-dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first-dimensional immobilized pH gradient gel strip prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evaluated. Silver stained two-dimensional patterns were obtained within 3.5 h.
TL;DR: The variety of two‐dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa.
Abstract: Holoprotein molecular weights and polypeptide composition can be determined for complex mixtures of oligomeric proteins using two-dimensional electrophoretic techniques. The variety of two-dimensional analyses presented here is a reflection of the general usefulness of each method for the identification and characterization of the different classes of seed storage proteins in alfalfa. These techniques can be applied to studies of storage proteins in other seeds as well as non-seed storage proteins. The major seed storage proteins in alfalfa are medicagin (a legumin-like globulin), alfin (a vicilin-like globulin) and a family of lower molecular weight albumins (LMW1-3). These comprise 30%, 10%, and 20%, respectively, of the total extractable protein from cotyledons of mature seeds. Alfin is a heterogeneous oligomeric protein (Mr approximately 150,000) composed of polypeptides ranging in size from Mr 14,000 to 50,000 (alpha 1-alpha 6; 50,000, 38,000, 32,000, 20,000, 16,000 and 14,000, respectively). Medicagin is also a high molecular weight oligomeric protein, but requires high concentrations of salt for solubilisation. It is comprised of a family of individually distinct subunits, each composed of an acidic polypeptide (A1-A9; Mr 49,000 to 39,000) linked via disulphide bond(s) to a basic polypeptide (B1, B2, B3; Mr 24,000, 23,000 and 20,000, respectively). This pairing is highly specific and two families are recognizable on the basis of the B polypeptide (B3 or B1/B2). Subunits (Mr approximately 50,000-65,000) are assembled as trimers (8S) or larger oligomers (12S-15S) in mature seeds. The lower molecular weight albumins (LMW1-3) are acidic (pI less than 6), and consist of sets of disulphide-bonded polypeptides (Mr 15,000 and 11,000).
TL;DR: Substitution of “strong” salts in the sample zone with salts formed by weak acids and bases essentially abolishes both phenomena, oxidation and irreversible denaturation of strong salts.
Abstract: Salts formed from strong acids and bases (e.g. NaCl, Na2SO4, Na2HPO4), present in a protein sample applied to an immobilized pH gradient (IPG) gel, induce protein modification (oxidation of iron moiety in hemoglobin) already at low levels (5 mM) and irreversible denaturation (precipitation) at higher levels (greater than 50 mM). This effect is due to production of strongly alkaline cationic and strongly acidic anionic boundaries formed by the splitting of the salt's ion constituents, as the protein zone is not and can not be buffered by the surrounding gel until it physically migrates into the gel matrix. Substitution of "strong" salts in the sample zone with salts formed by weak acids and bases, e.g.. Tris-acetate, Tris-glycinate, Good's buffers such as (N-[2-acetamido]-2-iminodiacetic acid (ADA), (2-[(2-amino-2-oxoethyl)-amino] ethanesulfonic acid (ACES), (3-[N-morpholino]propane sulfonic acid (MOPS), essentially abolishes both phenomena, oxidation and irreversible denaturation. Suppression of "strong" salt's effects is also achieved by adding, to the sample zone, carrier ampholytes in amounts proportional to the salt present (e.g. by maintaining a salt: carrier ampholytes molar ratio of at least 1:1). This suppression is due to the strong buffering power of the added carrier ampholytes, able to counteract drastic pH changes in the two moving boundaries. A reduction of these deleterious effects of strong salts is also achieved when the IPG run is performed at low voltage for a prolonged time (4 h at 500 V instead of only 1 h at 500 V, before switching to high-voltage settings). Guidelines are given for trouble-free IPG operations.
TL;DR: A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described, using ready‐made gels with three different pH intervals and pI marker proteins (Pharmacia).
Abstract: A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready-made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.
TL;DR: Two‐dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments of salt stress, water deficit, and osmotic stress on protein synthesis in roots of barley seedlings, finding that polypeptide changes induced by the stress treatments did not increase in response to water deficit or osmosis.
Abstract: Two-dimensional polyacrylamide gel electrophoresis was used to analyze and compare the effects of short term treatments (24 h) of salt stress, water deficit (desiccation), and osmotic stress (polyethylene glycol and mannitol) on protein synthesis in roots of barley seedlings (Hordeum vulgare L. cv. CM 72). These comparisons were made to determine if the polypeptides of Mr 26,000 and 27,000 and pI of 6.3 and 6.5 that were observed previously to increase significantly with salt stress (Plant Physiol. 1987, 83 517-524) also increased with water deficit and osmotic stress. The polypeptide patterns for control- and stress-treated plants were qualitatively similar, but the net synthesis of a number of polypeptides was quantitatively altered by each of the stress treatments. Of the polypeptide changes induced by the stress treatments, many were unique to a specific stress. Other polypeptide changes were common between two or more of the stress treatments. Only one polypeptide change, a decrease, was common to all of the stress treatments. An important finding was that polypeptides that increased significantly in response to salt stress did not increase in response to water deficit or osmotic stress.
TL;DR: By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non‐isotopic labeling and detection in routine parentage testing and Reproducible results were obtained.
Abstract: With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time-consuming. Most isotopic labeling techniques use the isotope 32P and require 3-10 days to visualize bands after hybridization. An alternative approach is based on the use of non-isotopic detection methods. The available non-isotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non-toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non-isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmid and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3-5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.
TL;DR: A new rapid imaging system based on a cooled charge‐coupled‐device was used to view the two‐dimensional fluorescent protein spot patterns and high resolution spot patterns were produced and compared with other methods of visualisation.
Abstract: A new method for visualising proteins in two-dimensional polyacrylamide gels was developed. Proteins were labelled with the fluorophore 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) while present in the first-dimensional gel after isoelectric focusing and subsequently electrophoresed into the second-dimensional gel. High resolution spot patterns were produced and compared with other methods of visualisation. A new rapid imaging system based on a cooled charge-coupled-device was used to view the two-dimensional fluorescent protein spot patterns. The method allows the immediate and rapid imaging of two-dimensional gels at the end of electrophoresis with no further processing.
TL;DR: The experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.
Abstract: Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e.g. 2-mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314-325). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e.g. more than 4 h at greater than or equal to 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2-mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2-ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.
TL;DR: A method allowing a clear separation of the different variants of desialylated alpha 1‐acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2 % v/v 2‐mercaptoethanol.
Abstract: A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.
TL;DR: A striking organ specificity was found by analyzing pleiotropic effect of gene substitutions in various organs of garden pea, revealing that according to the organ, the gene substitution caused appearances, disappearances or quantitative changes for 0 % to more than 10 % of the proteins revealed.
Abstract: Using high-resolution two-dimensional polyacrylamide gel electrophoresis we studied the polymorphism of protein amounts in some genotypes of maize and pea. This type of variability seems to be rather common and insensitive to environmental conditions, as attested by the comparison of the patterns of two maize lines harvested in two different years. A large-scale experiment involving 5 lines, 7 of their hybrids, and 6 organs (or physiological stages) of maize allowed us to examine numerous polypeptides regrading their genetic variability, their amount differences between organs and the inheritance of their abundance. Genetic and organ variations are not independent: polypeptides whose amount varies from one organ to another are, for the most part, genetically variable (59 %), while the stable polypeptides are not often genetically variable (18 %). We found a striking organ specificity for (i) the extent of quantitative variability (from 2.3–15.4 % of the polypetides), (ii) the occurrence and the type of variation for a given polypeptide (an intensity difference seen in an organ can disappear or even be reversed in another one), (iii) the kind of inheritance (additive/non-additive): combining the 6 organs and the 7 hybrids we found 101 cases of non-additivity (4 % of the total) which conecern as many as 72 different spots, that is to say that in most cases a polypeptide displaying nonadditivity in an organ seems to display additivity in the other ones. Moreover, for most of the polypeptides with nonadditive inheritance the hybrid spot presents an intensity similar to that of the most intense parental spot. Thus for protein experssion a hybrid cannot be predicted from its parents. The organ-specificity was also evidenced by analyzing pleiotropic effect of gene substitutions in various organs of garden pea. Comparisons of isogenic lines differing for the r-locus revealed that according to the organ, the gene substitution caused appearances, disappearances or quantitative changes for 0 % to more than 10 % of the proteins revealed. All these features are discussed in connection with the possible role of variation of protein amounts in phenotypic variability.
TL;DR: It is postulated that the measure of free mobility of the proteins is the M‐point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously, which abolishes the well‐known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo).
Abstract: In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2 %T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated μ-points) as their common free mobilities.
TL;DR: This new high‐pressure technique provides a direct means for studying quantitatively the effects of pressure upon protein‐ligand interactions and could become a suitable tool for the investigation of protein binding sites' topography.
Abstract: Hydrophobic affinity electrophoresis under high hydrostatic pressure has been developed to study the interaction between fatty acid-free bovine serum albumin and a long-chain aliphatic ligand physically immobilized within the gel matrix. From apparent association constants at various pressures and temperatures, apparent thermodynamic parameters including the volume change in binding were calculated. The results are as expected for hydrophobic interactions between the long-chain alkyl ligand and a high-affinity long-chain fatty acid binding site. The feasibility of high-pressure affinity electrophoresis is demonstrated. This new high-pressure technique provides a direct means for studying quantitatively the effects of pressure upon protein-ligand interactions. It could become a suitable tool for the investigation of protein binding sites' topography.
TL;DR: Three complementary two‐dimensional systems for the analysis of cereal prolamins are described and their effectiveness in analysing unreduced prolamin I fractions from wheat and rye is compared.
Abstract: Three complementary two-dimensional systems for the analysis of cereal prolamins are described. These are electrophoresis at pH 3.1 followed by electrophoresis at pH 9.2, isoelectric focusing (IEF) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and SDS-PAGE under non-reducing conditions followed by SDS-PAGE with reduction. They together give information on the pIs, Mrs and charge properties of the individual prolamin components, and on their interactions to form oligomers stabilized by inter-chain disulphide bonds. The three systems are then compared for their effectiveness in analysing unreduced prolamin I fractions from wheat and rye, and the IEF/SDS-PAGE system for analysing reduced and pyridylethylated prolamin fractions from all the major cereals. Finally, applications of the systems in biochemical and genetic studies are discussed and illustrated with three examples: analysis of the structural relationships of the S-rich prolamins (B hordeins and gamma-type hordeins) of barley, determination of the interactions of wheat and rye prolamin subunits in a 2RS/2BL translocation line, and the mapping of genes for alpha-type prolamins in the wild grass Haynaldia villosa.
TL;DR: Staining colloidal Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges.
Abstract: A new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5–10 mm with 0.1–0.2 % colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42 650) in 10 % w/v phosphoric acid. After staining for only 0.5–3 min, major zones, corresponding to 100–500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3 % w/v phosphoric acid for 5–80 min depending on gel thickness (120–500 μm) and type of support (fabric reinforced versus gels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1–2 ng/mm2 protein is found. Dye elution from stained fabric reinforced gels with 50 % v/v dioxane-water, followed by absorbance measurements results in a linear relationship over a range of 1–100 μg marker proteins. Staining colloidal Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the colloidal dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazadous.