bluuebunny/crossref_metadata_2025_split huggingface dataset:
A method of microcomputer‐aided two‐dimensional densitometry: An apparatus equipped with a chargecoupled device camera, and an algorithm of microcomputer programming
Authors: Toda Tosifusa , Fujita Toshiko , Ohashi Mochihiko 
Published: May 2005

AbstractA simple apparatus and a convenient program for evaluation of two‐dimensional electrophoretograms have been developed. The hardware system comprises a charge‐coupled device (CCD) camera, four pages of frame memory (FM), a page of display memory (DM), a video‐monitor cathode‐ray tube (CRT), a digitizer (DIG) and two ports of parallel interfaces (PIO). A small desk‐top microcomputer is connected to the apparatus via the interfaces. The system program named frame‐imagememory controller (FIMC) has been developed under an assembler of the microcomputer. FIMC is a package of subroutines, and utility programs for two‐dimensional densitometry can be written in BASIC language simply with the help of FIMC. Unevenness of basal gray level, caused by the nonuniformity of illumination in the camera's field of view, is eliminated from the pixel data by a method of blank subtraction. Background gray level, caused by protein staining procedures, is corrected by a method of local background subtraction. The two‐dimensional densitometry system, equipped with a CCD camera, is applicable not only to transparent gels but also to opaque membranes, and it shows a high reliability in optical measurement in the range of 0–2 O.D.
Improved blocking of nonspecific antibody binding sites on nitrocellulose membranes
Authors: Saravis Calvin A. 
Published: May 2005

AbstractNonspecific antibody binding either to nitrocellulose membrane sites or to transferred and immobilized proteins is eliminated (or minimized to insignificant levels) by treatment with a liquid gelatin preparation. This increases the specificity and sensitivity of nitrocellulose binding immunoassays, and allows incubation at 4°C which minimizes bacterial contamination and/or damage to biologically‐labile proteins.
Standardization of protein position in silver‐stained two‐dimensional polyacrylamide gel electrophoresis
Authors: Johnston Dennis A. , Capetillo Sylvia , Ramagli Louis S. , Guevara Juan , Gersten Douglas M. , Rodriguez Lewis V. 
Published: May 2005

AbstractMethods for standardization of silver‐“stained” protein position in two‐dimensional polyacrylamide gel electrophoresis are presented for their application to simultaneous multiple gel systems. Four methods are discussed: the use of a single gel per simultaneous run for standardization with either (1) known quantities of commercially prepared high‐or low‐molecular‐weight standards or (2) a known biological preparation such as T4 phage coat proteins; the use of each gel per simultaneous run to standardize itself by (3) including an internal carbamylation train such as creatine phosphokinase and using a one‐dimensional gel pattern of proteins such as rat heart whole homogenate along the margin(s) or (4) identifying “marker” protein spots in test samples that occur in all gels in a given study and standardizing to relative position. Method four is discussed in detail and examples given of its use in a comparative study of proteins in spontaneous primary hepatocellular carcinomas occurring in mice, and its use compared to the other methods.
Effects of alcohol abuse on human serum proteins revealed by two‐dimensional electrophoresis
Authors: Marshall Thomas , Vesterberg Olof , Williams Katherine M. 
Published: May 2005

AbstractAlcohol‐associated changes in human serum protein patrerns have been detected by silver staining following high resolution two‐dimensional electrophoresis and isoelectric focusing‐multiple gel piece electrophoresis. In the sera of alcoholics the concentration of α1‐acid glycorportein was consistently increased and in a high proporation of sera elevated levels of IgA, α1‐antichymotrypsin, haptoglobins and apo A‐I lipoprotein were observed. Increased levels of additional unidentified polypeptides were also detected. In contrast, reduced levels of antithrombin III were predominantly associated with the sera of alcoholics. The use of these polypeptides as potential clinical markers for alcohol abuse is discussed.
Phosphoglucomutase‐1 subtyping of human bloodstains on ultrathin‐layer polyacrylamide gels
Authors: Budowle Bruce 
Published: May 2005

AbstractA method is described for obtaining nondistorted, reproducible phosphoglucomutase‐1 subtyping patterns from fresh human bloodstains by ultrathin‐layer polyacrylamide gel isoelectric focusing. Electrofocusing of phosphoglucomutase on 0.2 mm thick gels took 52 and 80 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm, whereas a 9.5 cm distance between electrode wicks required 95 min on the Multiphor to complete isoelectric focusing. The gels with 8.0 cm between electrode wicks provided sharper band patterns compared with the gels with 9.5 cm between electrode wicks.
Increasing the sensitivity of protein detection of a silver stain for agarose gels
Authors: Budowle Bruce 
Published: May 2005

AbstractA silver staining method is described for increasing the sensitivity of protein detection in agarose gels. Following electrophoresis, the gels are first stained with Coomassie Brilliant Blue R‐250, then washed with glutaraldehyde and finally silver stained. Using this double‐stain method, protein detection was 100 times more sensitive than Coomassie Blue staining alone and 10 times more sensitive than when silver staining was used alone.
Double‐label autoradiography revisited: I. The 3H and 35S‐system
Authors: McConkey Edwin H. , Anderson Catherine 
Published: May 2005

AbstractThe method of double‐label autoradiography described by McConkey (Anal. Biochem. 1979, 96, 39‐‐44) has been updated for the use of the commercial flour. EN3HANCE, and the Lodak films XAR and SB. Fore gels 0.8 mm thick, the input ratio of 3h dpm/35S dpm should be 11.1 and the second‐stage authoradiogram should be exposed 5.9 × as long as the flurogram. For gels 0.4 mm thick, the input ratio of 3 dpm/ 35 dpm should be 5.3 and the second‐stage autoradiogram should be exposed 3.1 × as long as the fluorogram.
Swelling kinetics of immobiline gels for isoelectric focusing
Authors: Gelfi Cecilia , Righetti Pier Giorgio 
Published: May 2005

AbstractThe possibility of polymerizing Immobiline gels by a batch procedure and then storing them dry for subsequent use has been investigated. The gel swelling kinetics have been studied as a function of the following variables: a) solvent composition (plain water, 8 M urea, 2% Nonidet P‐40 and their mixture); b) gel composition (varying %T at fixed %C); c) temperature (in the range 20–60°C); d) gel thickness (from 0.5 to 2 mm); e) operative pH interval (from the extreme acidic, pH 3.5‐5, to the extreme alkaline, pH 8.5–10, ranges) under isoionic strength conditions. The following practical guide‐lines for gel drying should be observed: a) the gels should have a low matrix content (3 %T or lower); b) they should have reduced thickness (0.5, max. 1 mm); c) alkaline gels should be dried in vacuo over silica, to prevent CO2 absorption; d) the gels should be extensively washed prior to drying. Swelling kinetics are discussed in terms of Langmuir adsorption isotherms, while the diffusion of water in Immobiline gels does not seem to follow a passive Fickian mechanism.
Selective detection of calmodulin in polyacrylamide gels by double staining with Coomassie Blue and silver
Authors: Rochette‐Egly Cècile , Stussi‐Garaud Christiane 
Published: May 2005

AbstractIn the classical procedures following gel electrophoresis, calmodulin fails to stain with silver. Furthermore, once stained with Coomassie Brilliant Blue R‐250, calmodulinis not capable of undergoing silver staining and remains as a blue band except when pretreated with glutaraldehyde. Such a double stain technique allows the selctive detection of calmodulin in a mixture of proteins. However, it demonstrates some limitations of the silver staining procedures.
Effect of radioactive amino acids on chromosomes, viability and two‐dimensional protein patterns of cultured mammalian cells
Authors: Zeindl Evelyn , Klose Joachim 
Published: May 2005

AbstractThe effect of 14C‐labelled amino acids on three different established mammalian cell lines, i. e., on Hep‐cells, HeLa‐cells, and Chinese hamster ovary cells, was studied. The cells were incubated in the presence of 20 μCi 14C‐amino acids per 1 ml culture medium for 0–24 h or in the presence of 200 μCi/ml for 30 min. Thereafter, the chromosomes of the cells, the ability of the cells to form colonies (plating efficiency) and the protein patterns of the cells obtained by two‐dimensional electrophoresis were investigated. The results showed and increase in the number of chromosome breaks and a decrease in the plating efficiency when the incubation period was increased. In contrast, the frequency of qualitative and quantitative changes in the protein patterns of cells exposed to radioactive amino acids was not increased over the control level. Possible reasons for this discrepancy are discussed.
A method for dissociating group‐specific component from protein complexes in human bloodstains and its significance in forensic biology
Authors: Westwood Sara A. 
Published: May 2005

AbstractIn some bloodstains group‐specific component (Gc) forms complexes with blood platelet membrane proteins such as actin which, during isoelectric focusing (IEF), migrate with a lower isoelectric point (pI) than free Gc. When this association occurs it is difficult and sometimes impossible to identify the phenotypes of the Gc system. The problem has been overcome by extracting the bloodstain with 6 M urea prior to IEF. It is then possible to detect and to type Gc in bloodstains that would otherwise not be amenable to Gc typing.
Detection of transient and steady states in electrophoresis: Description and applications of a new apparatus with 255 potential gradient detectors along the separation trough
Authors: Thormann W. , Arn D. , Schumacher E. 
Published: May 2005

AbstractIntroduction of a linear of detectors along the electrophoretic separation space permits monitoring the evolution of electrophoretic zone structures, fully controlled by a microprocessor system. Both transient and steady state zone structures can be recognized. A description of our capillary type apparatus, with 255 potential gradient sensors measuring the most general physical property characterizing the separand pattern in electrophoresis, is given. The central unit of this device is a unique pattern of 256 electrodes. They are chemically photoetched on a supporting glass plate from a vapor‐deposited thin layer of a conductor. Each sensor has a width of 60 μm, a height of about 0.1μm and they are spaced 340 μm apart. The whole separation cell, appropriate detection electronics and a new constant current high tension power supply are described together with the microcomputer system used for control and data treatment. Illustrations of detected zone structures with steady state moving boundaries are given in terms of isotachophoretic and moving boundary experiments. The monitoring of non‐steady state boundaries is shown by following sample zones in zone electrophoresis.
Demonstration of proteins in electrophoresis gels using radioactive stains
Authors: Zapolski Edward J. , Podrasky Frank , Ledley Robert S. , Gersten Douglas M. 
Published: May 2005

AbstractWe describe the use of 35S‐labelled stains for the detection of submicrogram amounts of protein in SDS polyacrylamide gels. Many stains used to detect protein can be rendered radioactive, and proteins can be detected by radiofluorography due to stain binding in the absence of visible color. Of the stains tested. [35S]Ponceau Red S and [35S]Serva Blue R gave the best results. The potential utility of protein detection with radioactive stains is discussed.
Titration curves in ultrathin polyacrylamide and agarose gels
Authors: Gunzer Gerhard 
Published: May 2005

AbstractA technique for the preparation of ultrathin titration curve gels, 50 μm polyacrylamide gels and 100 μm agarose gels, is presented. By separating protein standard mixtures the advantages of this ultrathin‐layer technique in terms of more convenient handling, cost and time are demonstrated.
Ultrathin‐layer isoelectric focusing in agarose gels and visualization of urinary alanine aminopeptidase with a modified agar overlay technique
Authors: van Triet Adrianus J. 
Published: May 2005

AbstractUltrathin‐layer isoelectric focusing of the urinary alanine aminopeptidase in 100 μm agarose gels resulted in a better separation of the isoenzymes and proteins than in polyacrylamide gels. Although miniature isoelectric focusing over a 4 cm separation distance proved excellent for screening purposes, more details were observed in gels with a 10 cm separation distance. Optimum enzyme visualization was achieved with a modified overlay technique employing a 0.2% agar with substrate instead of the conventional 1 % agar overlay. After incubation the diluted agar overlay may be easily washed off without damaging the running gel.
Improved rehydration procedure for polyacrylamide gels in presence of urea: Demonstration of inherited human prealbumin variants by isoelectric focusing in an immobilized pH gradient
Authors: Altland Klaus , Banzhoff Angelika , Hackler Rolf , Rossmann Ute 
Published: May 2005

AbstractPolyacrylamide gels containing immobilized pH gradients (IPGs) are usually extensively washed in deionized water after polymerization and dried when longer storage is required. The original volume of the gel is readjusted by rehydration in water or a solution of appropriate composition and subsequent evaporation, if necessary, by using a fan for weight control. A more convenient procedure is to put the dried gels back into their casting mold used for polymerization and to fill the mold with the appropriate rehydrating solution. Rehydration to the original volume is completed within 30 min for gels of a thickness of 0.5 mm. With gels to be rehydrated in the presence of urea, it was found that water enters the gel matrix faster than urea; consequently, adjustment to the original gel volume at the required urea concentration is not possible in an excess of rehydration solution, but is easily achieved by the modified rehydration procedure. It was also possible to infuse a urea gradient into the gel matrix perpendicular to the pH gradient axis. The effect of urea on the patterns of inherited human prealbumin variants is demonstrated.
A silver stain for isoelectric focusing in agarose gel and its application for analyzing unconcentrated cerebrospinal fluid
Authors: Black Jeffrey A. 
Published: May 2005

AbstractA silver stain method has been developed for isoelectric focusing in agarose gel. Its sensitivity is shown to be greater than 20 times that of Coomassie Brilliant Blue G‐250. Its usefulness in analyzing unconcentrated cerebrospinal fluid is demonstrated.
An electroimmunoassay for sodium dodecyl sulfatesolubilized proteins in presence of the detergent
Authors: Fourcroy Pierre , Malfoy Bernard 
Published: May 2005

AbstractA procedure for the electroimmunoassay of sodium dodecyl sulfate (SDS)‐solubilized proteins is described. The analysis of the small subunit of ribulose 1,5‐bisphosphate carboxylase in antibody‐containing gels was best achieved in presence of low SDS concentration. Polyethylene glycol, low electroendosmosis agarose and medium voltage were required for optimized assay. Bovine serum albumin can also be analyzed in SDS/antibody gels. The results suggest that the electroimmunoassay in SDS‐containing gels could be used for many proteins.
Identification of cultivar‐specific proteins of winter wheat (T. aestivum L.) by high resolution two‐dimensional polyacrylamide gel electrophoresis and color‐based silver stain
Authors: Dunbar Bohn D. , Bundman Donnie S. , Dunbar Bonnie S. 
Published: May 2005

AbstractThe polypeptide patterns obtained from individual caryopses (kernels) of two wheat cultivars were analyzed using high resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) methods. While few polypeptides were detected with Coomassie Blue staining methods, over two hundred peptides were easily distinguished with the color‐based silver stain. The relative isoelectric points of the constituent polypeptides ranged from pH 3.8 to 9 and the relative molecular weights from 5000 to 200 000. The majority of the proteins of the two wheat varieties analyzed with 2‐D PAGE were similar although distinct proteins having molecular weights of 23 000, 30 000, 37 000 and 70 000 were identified, which have molecular properties unique to each variety. The use of the color‐based silver stain makes it possible to identify and characterize hundreds of proteins in individual wheat seeds. These methods are, therefore, adaptable for the rapid analysis and characterization of specific gene products of single kernels of wheat.
Protein separation in pH gradients using free‐flow electrophoretic apparatus I. The borate‐mannitol pH gradients
Authors: Shukun Sergey A. , Gavryushkin Alexander V. , Brezgunov Vyacheslav N. , Zav'yalov Vladimir P. 
Published: May 2005

AbstractProtein separation in borate‐mannitol pH gradients using a free‐flow electrophoretic apparatus was studied. The pH gradients covering 0.6–1 pH unit were created by dilution of mannitol solution in borax with distilled water. Linear concentration gradients of mannitol and borax, an almost linear pH gradient, and a linear conductivity gradient, increasing from the alkaline toward the acidic end of the pH gradient, were obtained. By varying the initial mannitol concentration in borax, the gradients can be formed within the pH range 4.5–9.2. Under the experimental conditions the gradients were rapidly changed due to the influence of the electric field. The character of changes was dependent on the properties of the membranes between the electrode compartments and separation chamber, on residence time, voltage and presence of salts in the gradients. Because of gradient instability, isoelectric focusing was not achieved under the experimental conditions, and the protein separation resulted from electrophoresis in the pH gradient. The results of the separation were dependent on the point of injection of the sample into the pH gradient: the best separation was achieved when the proteins were injected above their isoelectric points.
Protein separation in pH gradients using free‐flow electrophoretic apparatus II. The pH gradients formed by the concentration gradient of boric acid in solutions of borax and mannitol
Authors: Shukun Sergey A. , Gavryushkin Alexander V. , Brezgunov Vyacheslav N. , Zav'yalov Vlamdimir P. 
Published: May 2005

AbstractProtein separation was investigated in the free‐flow apparatus using pH gradients generated by a concentration gradient of boric acid in a solution of borax. The linear pH gradient covering the range pH 8.2–9.2 was obtained in a solution of 2.5 mM borax due to the formation of the logarithmic concentration gradient of 0–45 mM boric acid. These pH gradients lacked a conductivity gradient in contrast to borate mannitol pH gradients, described in the joint report. By addition of mannitol into the gradient, at constant concentration, the pH was shifted toward more acidic values in proportion to the amount of mannitol added, without changing the range and shape of the pH gradient. According to this procedure of gradient formation the pH gradients can be obtained within the range from pH 3.5 to 9.2. The gradients were rapidly decaying under the experimental conditions. Thus, isoelectric focusing of proteins could not be achieved, and the separation resulted from electrophoresis in the pH gradient. The results of protein separation in the gradients practically did not depend on the point of injection of the samples into the pH gradient, at least in the cases when the proteins were injected near their isoelectric points.
Transferrin subtypes determined by ultrathin‐layer polyacrylamide gel isoelectric focusing
Authors: Budowle Bruce 
Published: May 2005

AbstractAn ultrathin‐layer polyacrylamide gel isoelectric focusing method is described for subtyping transferrin. After a one‐hour pretreatment at room temperature of transferrin in serum with ferrous ammonium sulfate, the samples were subjected to isoelectric focusing for 140 min with a final voltage of 3500 V. Thus, transferrin subtyping was achieved in one‐half of a working day. Population data on transferrin in Black and White samples from Baltimore, Maryland, were obtained using this method. The data were similar to that previously reported on these races from other geographical locations.
Control of elecroendosmosis on agarose gellayers by use of modified gelatins
Authors: Shibata Futoshi , Ogita Zen‐ichi 
Published: May 2005

AbstractGelatins with various electric charges were added to agarose to control the strength of electroendosmotic flow and its effect on esterase zymograms was studied. Addition of the “basic fraction of gelatin” suppressed electroendosmotic flow. In contrast, addition of the “acidic fraction of gelatin” or “modified gelatin” intensified electroendosmotic flow. Modified gelatin was prepared by acylation of amino groups in the basic fraction of gelatin. The observed effects depended on the amount of gelatin added. The gelatin was shown to control electroendosmosis due to electric charge. In addition, the gelatin was found to exhibit a molecular sieving effect. Electroendosmotic flow and molecular sieving originating from the gelatin resulted in high resolution of the esterase zymograms.
Effects of ligand binding upon measurement of Gc by rocket immunoelectrophoresis: Implications for protein determination and for studies of protein/ligand interactions
Authors: Goldschmidt‐Clermont Pascal J. , Galbraith Robert M. , Emerson David L. , Nel Andre E. , Werner Philip A. M. , Lee William M. 
Published: May 2005

AbstractThe effect of protein complexing on quantitation by rocket immunoelectrophoresis was studied using group‐specific component (Gc), a serum protein which undergoes 1:1 interactions with both sterols (Vitamin D metabolites) and protein (G‐actin). Gc purified from serum was used as a reference. Complex formation with 25‐〈OH〉 D3 did not significantly affect the measurement of Gc. However, addition of excess G‐actin to purified Gc led to substantially higher rockets at every concentration of Gc tested. In dose‐response experiments performed with a constant quantity of Gc and increasing quantities of added G‐actin, the increment in rocket height was progressive up to equimolarity, at which point a plateau was reached. Further demonstration that the increased rocket heights were directly related to 1:1 complex formation between Gc and G‐actin (Gc:G‐actin) was obtained by [125I]G‐acting and isoelectric focusing (IEF) with autoradiography. Examination of the relative charge of Gc and of Gc:G‐actin by crossed immunoelectrophoresis showed that complexes exhibited faster mobility, whereas very little evidence of alteration in antigenicity of Gc:G‐actin complexes was apparent. These results suggest that Gc:G‐actin complexes exhibit increased electrophoretic mobility and that their presence in samples containing Gc causes an increase in the height of Gc rockets. The findings also indicate that interactions with specific ligands could cause artefacts in immunoelectrophorestic quantitation of other proteins present in biological fluids.