bluuebunny/crossref_metadata_2025_split huggingface dataset:
Observations and implications on the migration of calmodulin in a two‐dimensional gel system
Authors: Heydorn William E. , Creed G. Joseph , Jacobowitz David M. 
Published: May 2005

AbstractThese studies investigated the effect of calcium on the migration of calmodulin in a two‐dimensional (2‐D) gel system. As reported for one‐dimensional gels, calmodulin migrates on 2‐D gels primarily as a 21 kDa protein in the presence of 0.1 mM EDTA, and as a 15 kDa protein in the presence of 0.1 mM CaCl2. However, under basal conditions (i.e., neither EDTA nor calcium added to the bùffer system), calmodulin migrates on a 2‐D gel as a doublet of 20.3 and 18.7 kDa. These results suggest that sufficient residual calcium may be present in one (or more) of the standard laboratory reagents used in running 2‐D gels to produce this unique migration pattern for calmodulin. These results may also have implications for the migration of other proteins on 2‐D gels.
Gel electrophoresis with discontinuous rotation of the gel: An alternative to gel electrophoresis with changing direction of the electrical field
Authors: Serwer Philip 
Published: May 2005

AbstractWhen using agarose gel electrophoresis to fractionate linear DNA longer than 40–60 kilobases (kB), improved resolution by length has previously been obtained by periodically and discontinuously changing the direction the electrical field. This change of the field's direction was accomplished by alternately activating two sets of electrodes. However, some of the procedures used produce inhomogeneous electrical fields and with all of these procedures it is comparatively difficult to either change the angle between the two directions of the field or suppress formation of local pH gradients. To overcome these problems, a procedure is presented for changing the direction of the electrical field by rotating the gel. During agarose gel electrophoresis, a commercially available stepping motor and indexer are used to periodically rotate a gel on a circular disk within a conventional, horizontal, submerged gel electrophoresis apparatus. Because of the homogeneous field used, DNA forms comparatively sharp, undistorted bands. The previously achieved improved resolution of 40–166 kB, linear, double‐stranded DNÀ has been achieved here by rotating the gel. Dependence of length resolution on both the angle of rotation and the temperature has been measured.
Tetramethylurea as a protein denaturing agent in gel electrophoresis
Authors: Zazra James J. , Szklaruk Boris 
Published: May 2005

AbstractWe have examined the utility of tetramethylurea as a protein denaturant in electrophoresis using transverse gradient gels. Its physical properties make it easier to work with than urea and it appears to denature bovine serum albumin in a similar way. Tetramethylurea is a potential substitute for urea in some electrophoretic systems.
Alpha 1‐antitrypsin and orosomucoid phenotyping using allo A lectin‐peroxidase for detection
Authors: Umetsu Kazuo , Yuasa Isao , Taketa Kazuhisa , Suzuki Tsuneo 
Published: May 2005

AbstractA new method of identifying allo A lectin‐binding glycoproteins separated by isoelectric focusing is described. The method is based on the combination of neuraminidase treatment, isoelectric focusing and Durapore filter blotting followed by detection with allo A lectin. The technique is simple and reproducible for the determination of both the alpha 1‐antitrypsin and orosomucoid phenotypes in genetic and forensic investigations.
Evaluation of recombinant human tumor necrosis factor by cell electrophoresis. II. In vivo effect of recombinant human tumor necrosis factor on cell populations and natural killer‐activity in spleen of tumor‐bearing mice
Authors: Rychly Joachim , Knippel Eberhard , Milleck Jürgen , Schütt Wolfgang , Nizze Horst , Kramp Burghard , Klinkmann Horst 
Published: May 2005

AbstractThe effect of recombinant human tumor necrosis factor (rH‐TNF) on the composition and natural killer (NK)‐activity of lymphocytes in the spleen of normal and tumor‐bearing CBA mice was investigated. The characterization of cell composition was based on the analytical separation of splenic subpopulations differing in electrophoretic mobility. In untreated tunor mice, an increase of the electrophoretically intermediate population was found at the final stage of tumor growth. Treatment with TNF depressed this intermediate cell population in the spleen of tumorbearing mice, the electrophoretic distribution of splenocytes approaching the normal pattern. Also in normal mice TNF treatment affected the electrophoretic pattern. Already the first injection of TNF induced an increase of the intermediate cell population in the spleen. Tumor‐bearing mice showed a decreased NK‐activity at the final stage. In normal but not in tumor‐bearing mice repeated injections of TNF seemed to depress the NK‐activity. Cell electrophoresis represents a useful method for evaluating the effect of TNF on the cell populations in the spleen of mice.
A study of protein synthesis in subpopulations of cultured human epidermal keratinocytes using two‐dimensional gel electrophoresis
Authors: Easty David J. , Patel Ketan , Dover Robin , Dunn Michael J. , Evans David J. 
Published: May 2005

AbstractA procedure for the isolation of protein markers of epidermal differentiation in vitro is described. Human epidermal keratinocytes were cultured and radiolabelled in vitro. Fractionation was performed according to buoyant density (which reflects the degree of terminal differentiation) using Percoll density gradient centrifugation. Subpopulations of keratinocytes were characterised using light and electron microscopy, and proteins fractionated using high resolution two‐dimensional gel electrophoresis. Radio‐labelled proteins were detected using autoradiography and fluorography. Integral membrane proteins were characterised using Triton X‐114 phase shift extraction. Data from this in vitro study were compared to silver stained gels of samples from intact epidermis (in vivo). We report quantitative differences between 14 specific protein moieties expressed in subpopulations of keratinocytes and identify some of these proteins. The differential expression of these protein markers and their possible use in the interpretation of the keratinocyte maturation pathway in cultured cells from patients with skin diseases are discussed.
A comparison of rapid staining techniques for one‐ and two‐dimensional polyacrylamide gels
Authors: Patel Ketan , Easty David , Bailey Richard , Dunn Michael J. , Evans David 
Published: May 2005

AbstractTwo rapid staining procedures using 8‐anilino‐1‐naphthalene sulphonate (ANS) and Nitro Blue Tetrazolium (NBT) were compared with a Coomassie Brilliant Blue R‐250 staining technique. The methods were applied to protein separations using both one‐ (1‐D) and two‐dimensional (2‐D) polyacrylamide gel electrophoresis. The ANS procedure worked well for both 1‐D and 2‐D gels. The NBT technique, although more rapid, gave satisfactory results only for 2‐D gels. The ANS and NBT staining methods were both compatible with Western blotting so that they could be used for visualisation of the separation profile prior to blot transfer to nitrocellulose.
Electrophoretic and kinetic characterization of two phenotypes of soluble cytoplasmic L‐aspartate:2‐oxoglutarate aminotransferase in human liver tissues
Authors: Kanemitsu Fusae 
Published: May 2005

AbstractTwo phenotypes of soluble cytoplasmic L‐aspartate:2‐oxoglutarate aminotransferase (AST, EC 2.6.1.1), sAST 1 and sAST 2–1, were partially purified from human liver tissues and characterized by electrophoretic and kinetic analyses. sAST 1 had 2 bands upon electrophoresis on a Cellogel membrane, while sAST 2–1 had 3 bands with an additional anodic one. The frequencies of the two phenotypes of sASTs studied with 1,023 Japanese hemolysates were 98.5% for sAST 1 and 1.5% for sAST 2–1. sAST 1 was electrofocused to 9 bands with pI's ranging from pH 5.5 to 6.1 and sAST 2–1 into 11 bands with pI's ranging from pH 5.3 to 6.1. The molecular weights of the two phenotypes were both estimated to be 95 000. The apparent Km values of the sASTs for L‐aspartate with endogenous pyridoxal 5‐phosphate (PALP) were both 5.2 mM and those with added PALP were 5.9 mM for sAST 1 and 5.8 mM for sAST 2–1. The apparent Km values for 2‐oxoglutarate with endogenous PALP were both 0.26 mM and those with added PALP were 0.41 mM for sAST 1 and 0.37 mM for sAST 2–1. sAST 2–1 was more heat‐stable than sAST 1 upon incubation at 65 °C for up to 80 min, while both preparations were similarly denatured by treating with up to 4 M urea at 28 °C for 90 min. The two sASTs reacted with anti‐human AST‐IgG separated from AST‐IgG complexes in serum.
Fast horizontal electrophoresis. I. Isoelectric focusing and polyacrylamide gel electrophoresis using PhastSystem™
Authors: Olsson Ingmar , Axiö‐Fredriksson Ulla‐Britt , Degerman Margareta , Olsson Birgitta 
Published: May 2005

AbstractPhastSystem, 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 developement, 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.
Improved silver staining procedure for fast staining in PhastSystem Development Unit. I. Staining of sodium dodecyl sulfate gels
Authors: Heukeshoven Jochen , Dernick Rudolf 
Published: May 2005

AbstractA 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.
Horizontal two‐dimensional electrophoresis with immobilized pH gradients using PhastSystem
Authors: Görg Angelika , Postel Wilhelm , Günther Siegfried , Friedrich Claudia 
Published: May 2005

AbstractProtocols 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.
A continuous acetie acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines
Authors: Clements Robert L. 
Published: May 2005

AbstractA polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate‐lactic acid system (using 12 % acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.
Properties of thin‐rod immobilized pH gradients
Authors: Gianazza Elisabetta , Tedesco Giuliano , Cattò Nadia , Bontempi Licia , Righetti Pier Giorgio 
Published: May 2005

AbstractImmobilized pH gradient gel rods, 1.5 mm in diameter, were cast with a manifold connected to high‐precision burettes. The reproducibility of gel length was ca. 1.7 mm. The average standard deviation σx for spot position was 2 mm after one‐dimensional and 5.8 mm after two‐dimensional runs. In order to bring to completion the elution of the salt fronts into the electrode compartments, carrier ampholytes had to be included in the gel formulation at concentrations of at least 0.5–1%, depending on the pH range. The presence of carrier ampholytes, however, was troublesome in two respects: the gel tended to shrink and the cathodic bands drifted with time. Ionic components in the sample were tolerated up to the following concentrations: NaCl 8 μmoles, sodium dodecyl sulfate 10 μg per tube. In presence of non‐ionic detergents, the gels moved as a whole towards the cathode.
Orosomucoid 1 and orosomucoid 2 types in the Taiwanese and Japanese: Evidence for five new orosomucoid variants
Authors: Umetsu Kazuo , Yuasa Isao , Chen Eng‐Rin , Kudo Toshiyuki , Suzuki Tuneo 
Published: May 2005

AbstractThe genetic polymorphisms of orosomucoid, ORM1 and ORM2, were analyzed by isoelectric focusing on polyacrylamide gels and subsequent immunoprinting. Sera from 600 unrelated individuals in Taiwan and Japan were examined. Five new alleles, designated ORM1*7, ORM1*8, ORM1*4.2, ORM2*8 and ORM2*10, were observed together with common and rare alleles that have been found in the Japanese and the Filipinos. As compared with the Japanese, the Taiwanese have a higher frequency of ORM1*1, and a significantly lower frequency of ORM1*2·1.
Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G‐250 and R‐250
Authors: Neuhoff Volker , Arold Norbert , Taube Dieter , Ehrhardt Wolfgang 
Published: May 2005

AbstractAn 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 [1]. 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.
Ferguson plots based on absolute mobilities in polyacrylamide gel electrophoresis: Dependence of linearity of polymerization conditions and application to the determination of free mobility
Authors: Butterman Michael , Tietz Dietmar , Orbán László , Chrambach Andreas 
Published: May 2005

AbstractIn 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.
The use of biotinylated DNA probes in parentage testing: Non‐isotopic labeling and non‐toxic extraction
Authors: Dykes Dale D. 
Published: May 2005

AbstractWith 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 nonisotopic 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, plasmids 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.
Application of plasminogen polymorphism to forensic hemogenetics
Authors: Skoda Ulrike , Klein Anne , Lübcke Ingeborg , Mauff Gottfried , Pulverer Gerhard 
Published: May 2005

AbstractPlasminogen polymorphism (PLG) has attained considerable importance in forensic hemogenetics. PLG comprises two common, codominant autosomal alleles, PLG*A and PLG*B, more than 18 variants, and the silent allele PLG*QO. Isoelectric focusing followed by functional or immunochemical detection seems to be the optimal method for the determination of phenotypes. PLG*A is the most common allele in all populations, having its highest frequency in Mongoloids, Amerindians and Eskimos, the lowest in Caucasoids. The functionally inactive plasminogen M5 so far has been seen exclusively in Japanese individuals. Silent PLG alleles were only observe in the heterozygous state. No clear differences in functional activity or plasma level could be ascertained for any of the other allotypes. PLG polymorphism is now widely used for many haemogenetic investigations. From the allele distribution in European Caucasoids a single exclusion chance of 17.2 % for non‐fathers in paternity testing may be calculated. The major prerequisites of a new genetic marker in the parentage expertise, established Mendelian inheritance, favorable distribution of common alleles, low frequency of silent alleles, and simple reproducible typing technology, are fulfilled.
Subtyping of phosphoglucomutase 1 (PGM1) in microstains of different biological materials with subsequent species differentiation without further stain material consumption
Authors: Pflug Werner 
Published: May 2005

AbstractMicromethods for subtyping of phosphoglucomutase 1 (PGM 1) in small amounts of biological stain material are described, using an applicator for highly diluted stain extracts. With the aid of this applicator strip blood and semen micro‐stains as well as single hair‐roots could be extracted by electroelution directly on the PGM 1 isoelectric focusing gel. Species differentiation was also possible either by radial immunodiffusion using the extract remaining in the applicator strip after isoelectric focusing or by interpretation of the PGM pattern itself.
Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients
Authors: Kinzkofer‐Peresch Angelika , Patestos Nikos P. , Fauth Markus , Kögel Friedrich , Zok Roland , Radola Bertold J. 
Published: May 2005

AbstractAn optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5 %T, 3 %C, were internally stabilized with the aid of an AcrylAide‐pretreated, hydrophilized polyester fabric, preferably with a 60 μm mesh opening. Ultrathin (120–180 μm) gels were prepared with the flap technique and 500 μm gels with the cassette technique; 500 μm gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1–3 % w/v carrier ampholytes. Incorporation of 40–60 % w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900–1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1–0.2 μm pore size nitrocellulose membranes in 10–20 min from ultrathin gels and in 30–60 min from 500 μm gels. Based on quantification of residual protein in the gels after blottinǵ, a transfer efficiency of 60–87 % was found for the ultrathin and 53–69 % for the 500 μm gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris‐glycine buffer system electrophoretic blotting required only 2–5 min with ultrathin gels and 20 min with 500 μm gels. Marker proteins, including horse spleen ferritin (Mr465 000), could be transferred with 91–96 % efficiency.
Immunochemical detection of peptides and proteins on press‐blots after direct tissue gel isoelectric focusing
Authors: van der Sluis Paul J. , Pool Chris W. , Sluiter Arja A. 
Published: May 2005

AbstractA 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 μL 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 μm thickness. This allowed the detection of small peptides with a detection limit of approximately 10 pg/section.
Extraction of plant proteins for two‐dimensional electrophoresis
Authors: Granier Fabienne 
Published: May 2005

AbstractThree different extraction procedures for two‐dimensional electrophoresis of plant proteins are compared: (i) extraction of soluble proteins with a nondenaturing Trisbuffer, (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.