bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two‐dimensional electrophoresis of cereal prolamins: Applications to biochemical and genetic analyses
Authors: Shewry Peter R. , Parmar Saroj , Field J. Michael 
Published: May 2005

AbstractThree 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 γ‐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 α‐type prolamins in the wild grass Haynaldia villosa.
Electrophoretic heterogeneity of maize histones
Authors: Ivanchenko Maria G. , Georgieva Elena I. 
Published: May 2005

AbstractHistones from maize embryos and seedlings have been isolated using a fast extraction procedure. Three different electrophoretic systems have been applied for the study of the heterogeneity of maize core histones. Electrophoresis in acetic acid/urea polyacrylamide gels, containing high concentrations of urea, resulted in optimum fractionation of the core histones and especially of histone H4. Sodium dodecyl sulfate‐ containing polyacrylamide gels were not useful for the fractionation of maize histone classes H2a and H2b, nor for the various subfractions of H3 and H4. Gels containing Triton X‐100, used for the dimension in two‐dimensional electrophoresis proved to be efficient for the separation of all histone classes, as well as their structural variants and chemical modifications. Maize core histones have been oxidized in an attempt to define which of the Triton X‐100 resolved subfractions represent oxidation forms.
Routine diagnosis with PhastSystem compared to conventional electrophoresis: Automated sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, silver staining and Western blotting of urinary proteins
Authors: Scherberich Jürgen E. , Fischer Peter , Bigalke Annette , Stangl Pia , Wolf Gunter B. , Haimerl Marianne , Schoeppe Wilhelm 
Published: May 2005

AbstractThe recent introduction of the PhastSystem, an automatic electrophoresis and staining system with precast gradient‐gels, allows rapid and reproducible analysis of proteinuria in patients suffering from renal injury. A routine method for sodium dodecyl sulfate‐polyacrylamide gradient gel electrophoresis (SDS‐PAGE) and silver staining of unconcentrated urine specimens in the PhastSystem is described and compared to our conventional “macro”‐method with self‐cast SDS‐polyacrylamide gradient gels. The method described for the PhastSystem using 0.3 μL sample volumes and an 8–25% polyacrylamide gradient gel leads to highly reproducible results within 1.5 h. Before electrophoresis urine specimens were neither concentrated nor dialyzed. Samples with a protein concentration exceeding 5 mg/mL had to be diluted 1:5 (v/v). Analysis and documentation of PhastGels appeared as easy as with our conventional SDS‐PAGE. Protein bands could reliably be identified by Western blotting. Urine and serum proteins, separated in PhastGels, were electrophoretically transferred to nitrocellulose and detected with specific antibodies against human albumin, transferrin, alpha‐1‐antitrypsin and IgG. Comparison of several standard kits for molecular weight determination revealed considerable differences concerning the quality of protein separation patterns. Availability of precast gels and automatization of SDS‐PAGE and staining allows easy standarization of urine SDS‐PAGE among clinical routine laboratories.
Two‐dimensional electrophoretic analysis of human milk proteins
Authors: Goldfarb Marcia F. , Savadove Maureen S. , Inman Jean A. 
Published: May 2005

AbstractIdentification of human milk proteins and formulation of a two‐dimensional map is a first step in a project which intends to survey human milk proteins by two‐dimensional electrophoresis. Thirty‐four proteins have been identified using the Iso‐Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species‐specific, and, therefore, breast feeding confers a decided advantage for the infant. Using antisera for identification has revealed relationships between molecules which have not been previously noted. The antibody recognizes a common epitope between the IgA α chain and lactoferrin, and between the IgD d chain and β casein. Milk protein concentrations vary longitudinally, diurnally, and individually. Identification of the proteins contributes meaning to the varying patterns. Knowledge of human milk proteins will help to elucidate human nutrition and health needs.
Free flow electrophoresis as a method for the purification of enzymes from E. coli cell extract
Authors: Kuhn Reinhard , Wagner Horst 
Published: May 2005

AbstractThe application of the four techniques of free flow electrophoresis (zone electrophoresis, isotachophoresis, isoelectric focusing and field step electrophoresis) for the purification of proteins from a complex protein mixture was investigated. For this purpose α‐amylase (EC 3.2.1.1) from Aspergillus oryzae was added and reisolated from E. coli cell extract. The chosen enzyme and the biological extract are models for many industrial separation problems. In optimized experiments purity, purification factor, yield, throughput and efficiency were calculated. The best results were obtained with field step electrophoresis in combination with zone electrophoresis. High purity (0.82 mg enzyme/mg total protein) and high throughput (111 mL sample/h) were achieved using this technique. Field step electrophoresis gave the best throughput (330 mL sample/h), but low purity (0.63 mg enzyme/mg total protein). This technique can also be used for a simple concentration of the sample. With zone electrophoresis a purity of more than 0.95 mg enzyme/mg total protein was obtained, which was the best of all techniques. However, the enzyme concentration was decreased due to dilution with buffer solution after the separation. Isotachophoresis was the most difficult technique, combined with a relatively low recovery of 31% of the enzyme activity. In a purification scheme, free flow electrophoresis is able to substitute one or even several chromatography steps with a negligible loss of biological activity.
The microheterogeneity of serum α1‐antichymotrypsin revealed by interaction with concanavalin A in crossed immunoaffinoelectrophoresis and in affinity chromatography
Authors: Laine Anne , Hachulla Eric , Hayem Annette 
Published: May 2005

AbstractIn crossed immunoaffinoelectrophoresis with free concanavalin A in the first dimension, human serum α1‐antichymotrypsin, purified or in whole serum, exhibited four peaks in presence of 0.02 M α‐methyl‐D‐glucoside added to the second‐dimensional gel. α1‐Antichymotrypsin purified from the serum of a single healthy donor was separated by affinity chromatography into three fractions on a laboratory‐prepared concanavalin A‐Sepharose 4B column: a pass‐through fraction, a retarded fraction and a bound fraction, eluted from the column on addition of sugar to the buffer. These three fractions were analyzed by crossed immunoaffinoelectrophoresis and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and isoelectric focusing before and after desialylation. The results of electrophoresis as well as chemical analyses indicate that these microheterogeneous forms carry glycans with decreasing degrees of branching from the concanavalin A‐pass‐through form to the concanavalin A‐bound form. This approach represents a first step towards the elucidation of the molecular basis of the microheterogeneity of α1‐antichymotrypsin.
Study of human cerebrospinal fluid proteins by size exclusion‐high performance liquid chromatography and two‐dimensional gel electrophoresis
Authors: Wiederkehr Felix , Büeler Martin R. , Wacker Marianne , Vonderschmitt Dieter J. 
Published: May 2005

AbstractCerebrospinal fluid (CSF) proteins were separated into three main fractions by size exclusion‐high performance liquid chromatography (SE‐HPLC). Subsequent analysis of each fraction by two‐dimensional gel electrophoresis (2‐DE) facilitated the detection of trace components in CSF and additionally provided more information about the native properties of various proteins. Certain proteins are present in a polymeric form and appear in the high molecular weight SE‐HPLC fraction. In the middle molecular weight SE‐HPLC fraction we found a CSF‐specific transthyretinrelated protein by immunoblotting with polyclonal antibodies to transthyretin. Possible interpolypeptide disulfide bonds of such polymeric proteins were studied using a nonreducing 2‐DE system. This procedure revealed that all apolipoprotein E monomers in CSF, which are synthesized in astrocytes, are linked by disulfide bonds. In the CSF from a patient with clinically definite multiple sclerosis (MS), novel proteins appeared in the high molecular weight SE‐HPLC fraction, which are obscured by other proteins if total CSF is analyzed.
Detection of β‐ 1,3‐glucanase activity after native polyacrylamide gel electrophoresis: Application to tobacco pathogenesis‐related proteins
Authors: Côté Francois , Letarte Jocelyne , Grenier Jean , Trudel Jean , Asselin Alain 
Published: May 2005

Abstractβ‐1,3‐Glucanase (laminarinase) activity was detected after polyacrylamide gel electrophoresis under native conditions by using laminarin as substrate. Following incubation of gels, laminarin was stained with Aniline Blue. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. As low as 0.001 unit of commercial Penicillium laminarinase could be observed after incubating the polyacrylamide gel for 45 min at pH 5.0. Extracts of commercial Penicillium laminarinase exhibited four bands with lytic activity towards laminarin. Analysis of intercellular fluid extracts of tobacco mosaic virus‐infected tobacco leaves revealed four β‐1,3‐glucanases corresponding to three acidic pathogenesis‐related proteins, b4 (2), b5 (N) and b6b (0), and one basic protein. The presence of laminarin in gels retarded the migration of some proteins with β1, 3‐glucanase activity. This change in electrophoretic mobility could be used as a complementary affinity test for identifying proteins with β‐1,3‐glucanase activity.
Affinity electrophoresis for diagnosis fo cancer and inflammatory conditions
Authors: Brȩlorowicz Jan , Mackiewicz Andrzej 
Published: May 2005

AbstractAffinity electrophoresis, with concanavalin A and Lens culinaris agglutinin as ligands, was applied to study the microheterogeneity of serum proteins, with special emphasis on α1‐fetoprotein and α1‐acid glycoprotein, two proteins of potential clinical value. A total of 602 samples of serum from patients with various neoplastic and inflammatory conditions were evaluated. Affinity electrophoresis provided useful information for differential diagnosis of cancers of various origins including hepatomas, metastatic liver cancers and yolk sac tumors. The method also proved indispensable for the detection of intercurrent infection in patients with rheumatoid arthritis, systemic lupus erythematosus and scaled burns.
Two‐Dimensional electrophoresis of human serum proteins following acute myocardial infarction
Authors: Marshall Thomas , Williams John , Williams Katherine M. 
Published: May 2005

AbstractSerum proteins associated with acute myocardial infarction (AMI) have been monitored by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and high resolution two‐dimensional electrophoresis (2‐DE) under nonreducing conditions. Proteins a, b, c (Mr 13 000; pI 6.2, 6.7 and 7.5, respectively) and e (Mr 27 000; pI 5.2) appear simultaneously ∼ 30 h after infarction, reach maximum intensity after 48 h and progressively decline thereafter. Protein d (Mr 15 000; pI 7–8.5; identified as hemoglobin) sometimes appears within 18 h of infarction. Proteins a‐c are not detected in the 2‐DE patterns of healthy myocardium, infarcted myocardium, pectoral muscle or tongue, but e is present in all and tentatively identified as myosin light chain. Other myocardial proteins which are either reduced in amount following infarction or more specifically associated with myocardium than pectoral muscle are not detected in the serum of AMI patients. Analysis of unconcentrated urine by SDS‐PAGE and silver staining does not reveal proteins specific to AMI.
The impact of boundary conditions in free flow field step electrophoresis
Authors: Heydt Ariane , Mosher Richard A. 
Published: May 2005

AbstractThe impact of the type of membrane used to separate the electrolyte compartments from the separation cell for continuous flow field step electrophoresis is examined using both experimental and computer simulation data. It is shown that the distinct transport characteristics of ion exchange membranes and dialysis membranes may have an effect on the stability of the pH and conductivity gradients created by the buffer system. These effects are interpreted using computer simulation data. The limitations of the model used to portray the transport characteristics of ion exchange membranes are discussed as are the experimental conditions which provide the greatest stability for field step electrophoresis.
The electric field dependence of DNA mobilities in agarose gels: A reinvestigation
Authors: Holmes Diana L. , Stellwagen Nancy C. 
Published: May 2005

AbstractThe electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments ≤ 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained ≤ 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris‐borate buffer (TBE) than in gels cast and run in Tris‐acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incroporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature‐corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments ≤ 12 kbp in size in agarose gels ≤ 1.4 % in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments ≤ 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments ≥ 1 kbp in size in agarose gels ≥ 2 % in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an “intrinsic” DNA mobility of 2.7 x 10−4 cm2/Vs at 20°C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments. If the volume occupied by each DNA molecule is approximated by its root‐mean‐square (rms) radius of gyration, the median pore radius, rp, of an LE agarose gel cast and run in TAE buffer is rp = 98 A−0.90, where A is the agarose concentration in % and rp is given in nm. For gels cast and run in TBE buffer, the apparent median pore radius is rp = 66 A−0.95. If the volume occupied by each DNA molecule is represented by its mean geometric radius instead of the rms radius of gyration, the median gel pore radius in both buffers depends on A−0.5, as predicted by the extended Ogston theory. From the corresponding retardation coefficients, the gel fiber parameters were estimated.
Rapid detection of proteins by enzyme‐linked immunofiltration assay after transfer onto nitrocellulose membranes
Authors: Pinon Jean‐Michel , Puygauthier‐Toubas Dominique , Lepan Hervé , Marx Cathy , Bonhomme Annie , Boulant Joselyne , Geers RÉGine , Dupont Hervé 
Published: May 2005

AbstractEnzyme‐linked immunofiltration assay (ELIFA) for labeling transferred proteins is an interesting and powerful technique for the rapid specific detection (15 min) of proteins immobilized on nitrocellulose or nylon membranes (0.20 and 0.45 μm). ELIFA does not require fastidious handling of the membranes. Saturation, specific labeling and washing procedures are achieved by filtration, controlled by a monitoring unit which regulates the flow rate and ensures excellent specificity, repetition and reproducibility. The recycling by closed circuit or by repetetive inversion of the flow direction offers the advantage of reducing the volumes of expensive reagents while simultaneously increasing the sensitivity of the technique. The detection limit is at least as low as 1–5 ng using directly or indirectly enzymatically labelled probes. ELIFA may be extended to the identification of glycoproteins using specific ligands such as lectins or to the immunocapture of an antigen using specific antibodies immobilized on an activated membrane. ELIFA complements fast separation, by e.g., isoelectric focusing, polyacrylamide gel electrophoresis, or sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and accelerated electrotransfer to membranes with rapid detection reducing the total time for separation transfer and detection to less than 2 h.
A procedure for the extraction and high resolution two‐dimensional gel electrophoresis of total nuclear phosphoproteins from isotonically purified nuclei
Authors: Baciu Peter C. , Durham John P. 
Published: May 2005

AbstractMethods are described for the extraction and preparation of total nuclear proteins for high resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), The conditions for protein extraction and preparation limit both protease and phosphatase activity, allowing application of this technique to the reliable analysis of changes in nuclear protein composition and nuclear protein phosphorylation as well as other forms of post‐translational modifications. Unlike other procedures for 2‐D PAGE analysis of nuclear proteins the technique allows solublization and extraction of all nuclear proteins along with removal of nucleic acids which interfere with isoelectric focusing and autoradiography of 32Pi‐labeled proteins. It avoids lengthy dialysis in which precipitation of nuclear proteins often occurs and does not require precipitation and resolubilization of nuclear proteins to obtain sufficient protein concentrations for 2‐D PAGE analysis; often impractical steps in which complete resolubilization of all proteins is not possible. It produces high resolution 2‐D PAGE analysis in which identification of even low abundance proteins can be made, based on isoelectric point and molecular weight, allowing comparison with other studies.
Giant two‐dimensional gel electrophoresis: Methodological update and comparison with intermediate‐format gel systems
Authors: Levenson Richard M. , Anderson George M. , Cohn Jonathan A. , Blackshear Perry J. 
Published: May 2005

AbstractTwo‐dimensional (2‐D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970′s. Nevertheless, 2‐D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the „giantgel”︁ format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2‐D gel system available. As originally described, this procedure has several important limitations: it requires custom‐built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain gaint gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2‐D gel analyses. The smaller format gels resolved fewer proteins, by 30–40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in selected applications.
A plant dye from Lawsonia inermis for protein staining after polyacrylamide gel electrophoresis
Authors: Ali Rashda , Sayeed Syed Asad 
Published: May 2005

AbstractA reddish‐brown dye was isolated from the leaves of Lawsonia inermis by extraction with calcium hydroxide (pH 11–12). A 3.6% crude extract in ethanol/water, 1:1 v/v, was used for direct staining, without fixation, of bovine serum albumin, casein and human serum proteins, following polyacrylamide gel electrophoresis in cylindrical gels. After staining for 30 min the gels were destained for 1/2–2h with 7% acetic acid at 60°C. Protein staining with Amido Black 10B and Coomassie Brilliant Blue R‐250, according to standard protocols, required destaining for 24 h and more to obtain a comparably cleared background. Staining with the plant dye and Coomassie Brilliant Blue had a similar overall staining sensitivity but some minor components of human serum showed different staining characteristics with each of the two dyes. Staining with the plant dye excels over standard staining by speed and simplicity.