bluuebunny/crossref_metadata_2025 huggingface dataset:
Distribution of plasminogen allotypes in eight populations of the Western Hemisphere
Authors: Dykes Dale , Nelson Mary , Polesky Herbert
Published: May 2005

Phenotyping for plasminogen (PLG) variants on 0.5 mm agarose isoelectric focusing gels enabled us to identify six different allotypes among the eight populations studied. PLG *1 and PLG *2 were seen at polymorphic frequencies in all the populations studied except Eskimos which had a gene frequency of 0.993 for PLG *1. The variants PLG *3, PLG *B, were seen at low frequencies in some of the populations, whereas PLG *M was only identified in the local White population. A new variant named PLG *D, which we consider to be a marker of black populations, was identified in local Blacks and Black Caribs at frequencies of 0.037 and 0.058, respectively.
High resolution comparison of Plasmodium knowlesi clones of different variant antigen phenotypes by two‐dimensional gel electrophoresis and computer analysis
Authors: Howard Russel J. , Aley Stephen B. , Lemkin Peter F.
Published: May 2005

Antigenic variation of the schizont‐infected cell agglutination‐antigen on erythrocytes infected by Plasmodium knowlesi is due to the expression of different malarial proteins. Here we examined whether antigenic variation is accompanied by multiple phenotypic changes in the proteins synthesized by P. knowlesi. The malarial proteins of two clones, one of which was produced by antigenic variation from the other clone in vivo, were labeled by [3H]isoleucine uptake during in vitro parasite growth. The proteins were separated by two‐dimensional gel electrophoresis, detected by fluorography and compared using the GELLAB computer system. In the molecular weight range 35 000 to 230 000 and pH range 4.55 to 6.10, we found only four qualitative differences and two robust quantitative differences among approximately 500 proteins characterizing the protein phenotype of these clones. One clone exhibited four minor protein spots absent from the other. We conclude that antigenic variation in P. knowlesi represents the differential expression of a very small number of malarial genes.
A simple method for drying polyacrylamide slab gels using glycerol and gelatin
Authors: Popescu Octavian
Published: May 2005

A method for drying polyacrylamide slab gels of various size and gel composition (T 7.5–15 %, C 2.67 %) is described. The destained gels, containing glycerol, are overlayered with gelatin and dried. Transparent and flexible sheets are obtained which can be stored for extended periods and may also be used for autoradiography.
Detection of endoproteinases in polyacrylamide gels
Authors: Petersen Gary R. , Van Etten James L.
Published: May 2005

A simple and versatile method was developed for detecting endoproteinases by nondenaturing polyacrylamide gel electrophoresis. A vertical strip of the resolving gel is subjected horizontally to a second electrophoresis in the presence of a substrate protein and sodium dodecyl sulfate (SDS). The location of proteolytic activity in the original gel strip is revealed by the disappearance of substrate and the appearance of proteolytic products (due to limited proteolysis) in the second gel. The pattern of peptide fragments produced permits the identification of the protease responsible.
Molecular size and charge characterization by rheophoresis I. Theory and gel calibration
Authors: Waldmann‐Meyer Henrik , Pedersen Per Amstrup
Published: May 2005

Rheophoresis, i.e. thin‐layer electrophoresis in particulate gels with controlled evaporation, combines in one experiment the chromatographic and the electrophoretic characteristics of macromlecules in such a way that they can be determined independently of each other. In this paper theory and experimental corroboration for molecular size determination are presented. We describe the migration velocity of any molecule as the sum of an electrophoretic, an electroosmotic and a rheophoretic component. The latter is the velocity originating from the liquid flow produced by evaporation. It is a function of the position along the gel axis and the cross‐section available to the molecule, but totally independent of the molecular charge. As a result of the position dependence, the rheophoretic component can be isolated from the two others and the available cross‐section determined by means of a simple plot. Thus, the chromatographic partition coefficient is readily calculated and the effective hydrodynamic radius of the molecule obtained.
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

A 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

Nonspecific 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

Methods 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

Alcohol‐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

A 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

A 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

The 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

The 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

In 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

The 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

In 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

Introduction 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

We 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

A 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

Ultrathin‐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

Polyacrylamide 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

A 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

A 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

The 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

Protein 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

Protein 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

An 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

Gelatins 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

The 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.
Electrophoretic characterization of esterase‐19 (ES‐19), a new arylesterase of the house mouse (Mus musculus)
Authors: Münz Michael , von Deimling Otto
Published: May 2005

Esterase‐19 (ES‐19) of the house mouse, following specific visualization in electrophoretic and isoelectric focusing gels, is described. A list of diagnostic features was compiled including substrate specificity, inhibition and activation characteristics, temporal and spatial patterns of expression, distribution among laboratory and wild mouse strains. ES‐19 was classified as arylesterase (E.C. 3.1.1.2) and compared with ES‐16 and ES‐25, two other mouse arylesterases. Homology with brain esterases of rat, guinea pig, rabbit and man was found, and a uniform disegnation “arylesterase I” is proposed.
Reducing background interference in silver stained two‐dimensional polyacrylamide gels
Authors: Amess Robert , Fox John E. , Spragg Peter S.
Published: May 2005

Amino acid analysis shows that commercial grades of agarose contain peptides. The quantity varies with the purity of the agarose. These findings agree with our observations that the extent of the background after silver‐staining for agarose gels or where agarose was used in the system (as in two‐dimensional polyacrylamide gel electrophoresis) was determined mainly by the quality of agarose used.
Rapid mechanical versus conventional acid fixation in electrophoresis
Authors: Gersten Douglas M. , Zapolski Edward J. , Ledley Robert S.
Published: May 2005

We have devised methodology which allows for the rapid mechanical fixation of proteins in polyacrylamide slab gels. Proteins so fixed remain in the gel even after four days of soaking. The potential utility of this approach is discussed.
Induction of a mitochondrial matrix protein in human fibroblasts by interferon or poly I:C
Authors: Leigh Anderson N.
Published: May 2005

The abundance of protein Mitcon: 5, a major component of monocytes and granulocytes as observed by high‐resolution two‐dimensional electrophoresis, is increased in human fibrolasts treated with interferon or poly I:C. Production of this polypeptide is eliminated in cells treated with dinitrophenol, but not in cells treated with chloramphenicol, indicating that it is a cytoplasmically synthesized mitochondrial protein. Neither chloroquine nor tunicamycin, both of which interfere with proper targeting of lysozomal enzymes, affects Mitcon: 5 production. Actinomycin D prevents induction, indicating that new transcription is required. Cell fractionation studies show that Mitcon: 5 is found in the mitochondria/lysosome fraction, and that it is released from this fraction by treatments designed to liberate matrix enzymes. Mitcon: 5 thus appears to be the first example of a direct effect of interferon treatment on the composition of mitochondria. Furher characterization of this effect may help to explain side‐effects of interferon treatment, including fever.
A simplified procedure for evaluation and storage of isoelectric focusing gels prior to second‐dimension electrophoresis
Authors: Gower David J. , Tytell Michael
Published: May 2005

A simple method of stain fixation of polyacrylamide isoelectric focusing tube gels is described. Gel handling and storage are simplified by fixation and second‐dimension quality is markedly improved over that from the original procedure involving quick freezing and frozen storage of the gel.
Relationship between electrophoretic mobility and activation of human monocytes
Authors: Bauer Johann , Hannig Kurt
Published: May 2005

Isolated human monocytes showed an increased electrophoretic mobility (EM) during incubation in vitro. However, when the monocytes were isolated after activation in a mixed leukocyte culture, they tended to maintain their original EM during culturing. Activated and non‐activated monocytes were compared with respect to their activities during lymphocyte transformation. The lymphocytes which were used to test the activities of both kinds of monocytes had been depleted of all accessory cells by a two‐step physical isolation procedure including coutercurrent centrifugal elutriation and cell electrophoresis. Non‐activated monocytes supported concanavalin A (Con A) ‐ induced transformation of the purified lymphocytes optimally when the culture medium was supplemented by fetal calf serum (FCS). If, however, activated monocytes were used in lymphocyte transformation tests, Con A induced optimal [14C]thymine‐deoxyribose incorporation, even if FCS was replaced by autologous human plasma. The results suggest that there is a relationship between the stability of the EM of human monocytes and their ability to support lymphocyte proliferation during an in vivo immune response, when FCS is obviously not present.
Serum protein analysis on immobilized pH gradients with in situ adsorption of albumin on Dextran Blue
Authors: Gianazza Elisabetta , Giacon Patrizia , Astrua‐Testori Silvia , Righetti Pier Giorgio
Published: May 2005

Dextran Blue incorporated in concentrations up to 1% w/v into a gel layer buffered with Immobiline chemicals to pH 9.1 does not interfere with isoelectric focusing within an adjacent immobilized pH gradient (IPG) and retains its ability to strongly bind human serum albumin. With minor modifications of the procedure, it is possible to use Dextran Blue shelves in connection with either non‐denaturing or denaturing (i. e. containing 8 M urea) analysis media. Examples include easier typing of group‐specific component in an ultranarrow IPG against a completely clear background and improved resolution of transferrin and hemopexin in two‐dimensional separations.
Detection of non‐heme iron proteins following polyacrylamide gel electrophoresis
Authors: Ohmori Daijiro , Tanaka Yukiko , Yamakura Fumiyuki , Suzuki Koji
Published: May 2005

Non‐heme iron proteins were stainned in the gel with bathophenanthroline disulfonate after polyacrylamide gel electrophoresis. This staining method proved more resistant to color fading than iron detection by α,α′‐dipyridyl, with >50% of the color being retained after 24 h by three ferredoxins of different origin. A linear relationship was found for the amount of iron in non‐heme iron proteins, applied to the gel, and the areas of densitometric tracings.
A combination of Coomassie Blue and concanavalin A ‐ based glycoprotein stains avoids ambiguities in the assignment of bands
Authors: Spatz Hanns‐Christof , Waehneldt Thomas V. , Neuhoff Volker
Published: May 2005

A simple procedure is described to double‐stain sodium dodecyl sulfate‐polyacrylamide gels both with Coomassie Blue and by the concanavalin A ‐ horseradish peroxidase method to detect glycoproteins among the protein bands. Double staining proves useful in cases where many bands are present on the gels and where a comparison of banding patterns from separate gels or even separate lanes of the same gel cannot be carried out with sufficient accuracy.
Sensitive Nitro Blue Tetrazolium staining of proteins in high resolution two‐dimensional gel electrophoresis
Authors: Bahrman Nasser , Thiellement Hervé
Published: May 2005

A new method is described for staining proteins after high resolution two‐dimensional gel electrophoresis. The polyacrylamide gel matrix is stained blue, by photoreduction of Nitro Blue Tetrazolium, while the spots of polypeptides remain unstained. The method is much more sensitive than Coomassie Blue staining and its sensitivity is close to that obtained by silver staining.
The effects of high salt concentrations in the samples on molecular weight determination in sodium dodecyl sulfate polyacrylamide gel electrophoresis
Authors: See Yew Phew , Olley Peter M. , Jackowski George
Published: May 2005

For proper molecular weight determination in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), the protein should bind the same amount of SDS. High ionic strength had previously been shown to reduce the amount of SDS bound to proteins, suggesting that high salt concentrations in the samples may interfere with molecular weight determination. We examined the effects of NaCl from 0 to 1 M in the samples on molecular weight determination of proteins in SDS‐PAGE. In our conditions of electrophoresis, the retardation coefficients (KR), free mobilities (Yo) and Rf values for proteins (Mr 20 000–66 000) are not affected by NaCl in the samples up to 0.8 M. Therefore molecular weight of these proteins can be determined by both the Ferguson plot and Rf vs. log Mr methods in the presence of NaCl in the samples up to 0.8 M and compared with standard protein samples in the absence of NaCl. The Rf, KR and Yo values for phosphorylase (Mr 97 400) are affected by NaCl from 0.4 M or higher in the samples. Preliminary results show that increasing sodium phosphate, pH 7.0 from 0.01 M to 0.1 M in the sample affect molecular weight determinations. The effects of phosphate are greater in the Ferguson plot than in the Rfvs. log Mr plot procedure. In the latter method, the effects are dependent on the acrylamide gel concentrations and on the size of the proteins. We conclude that the molecular weight of proteins (Mr 20 000 to 97 000) can be determined in the presence of high salt concentration in the sample if the unknown and standard protein samples contain the same amount of salt and buffer ions.
Improved classification of alpha‐1‐antitrypsin in immobilized pH gradients containing sucrose
Authors: Pascali Vincenzo L. , Conte Gabriella
Published: May 2005

Two narrow immobilized pH gradients (pH 4.35–4.85 and pH 4.40–4.80) were formulated by linear interpolation from standard 1 pH unit ranges. Both gradients proved excellent for alpha‐1‐antitrypsin phenotype separation. Gel casting was simplified and the properties of the gels during the run improved by replacing glycerol with sucrose in the polymerization solutions.
Electrophoretic transport equations: Electrophoretic models based on migration only and their interrelationships
Authors: Thormann Wolfgang , Mosher Richard A.
Published: May 2005

The general equations which describe the electrophoretic transport of components in solution are restated using Newman's general concept of mobilities. A concise derivation of the moving boundary equation and the regulating function ω from the continuity equation is presented. Various other regulating principles across moving and stationary boundaries are also discussed, which permits a review of the features and interrelationshpis of the electrophoretic models based on electromigration only. The effect of considering an interactive (dissociating) solvent on the mathematical treatment is discussed.
Two‐dimensional fractionation of some particular human DNA restriction fragments
Authors: Guérin Patrick , Lucotte Gérard
Published: May 2005

A procedure which allows the resolution of a portion of the human genome into discrete bands corresponding to particular DNA fragments is described. Genomic DNA has been digested with a restriction endonuclease, and the resulting DNA fragments are fractionated according to size on a polyacrylamide gel. After elution of DNA each fraction is digested with a second restriction endonuclease and analysed after radioactive labeling on a second polyacrylamide gel. Autoradiograms of these gels reveal numerous bands with sizes below two kilobases. A reproducible pattern of bands between individuals tissues and species, is generated; most of them correspond to multicopy sequences, being cut simultaneously by these two particular restriction enzymes.
A discussion of the constraints which lead to steady state electrophoretic boundaries
Authors: Mosher Richard A. , Thormann Wolfgang
Published: May 2005

The electric field variation across moving boundaries in three component systems is analyzed with a model based on electromigration only (moving boundary equation and regulating function ω). A decrease in the electric field strength across a boundary in the direction of migration is the most general requirement for the establishment of steady state migrating boundaries. The formation of that electric field gradient is shown to be based on two fundamental constraints. These are (i) the higher net mobility of the leading component compared to that of the terminating constituent and (ii) the higher concentration ratio of the leading component to the terminating component ahead of the moving boundary compared to that in the terminating zone. The relations are discussed with respect to strong and weak electrolytes where all three components are present on either side of the boundary. The predictions are verified experimentally. Limitations of the model based on migration only are discussed.
Concentration of cerebrospinal fluid does not affect immunoglobulin G oligoclonal banding patterns
Authors: Shapshak Paul , Tourtellotte Wallace W. , Lee Mike M. , Staugaitis Susan M. , Cowan Tina , Ingram Tim
Published: May 2005

The effects of concentration on immunoglobulin G (IgG) oligoclonal bands after isoelectric focusing in cerebrospinal fluids (CSF) were examined using six concentration procedures, and the percent recoveries of IgG varied. The procedures, % IgG recoveries, and corresponding degrees of concentration are as follows: (1) Amicon CF25 membrane cone, 75 %, 45 ×; (2) Amicon CF50A membrane cone, 65 %, 3 ×; (3) Amicon CS15 Minicon, 47 %, 80 ×; (4) Microprodicon, 55 %, 35 ×; (5) collodion bag negative pressure dialysis, 50 %, 16 ×; and (6) microcollodion bag in Permasorb, 40–80 %, 10–60 ×. Ultracentrifugation of CSF at 100 000 g for 1 h prior to concentration using CF 25 membrane cones introduced not more than 5 % variation in the IgG concentrations. Isoelectric focusing was performed on 20 CSF samples concentrated by CF25 membrane cone, CS15 Minicon, and collodion bag, and no effects on the oligoclonal IgG banding patterns were found.
Background estimation in one‐dimensional electropherograms of whole‐cell protein extracts
Authors: Lasters Ignace , Leyns Frederik , Jackman Peter J. H.
Published: May 2005

In electropherograms of whole‐cell protein extracts a background absorbance due to many partially overlapping bands is usually observed. Since this background is in general not relevant for the identification of a given pattern, its removal will increase the discrimination between patterns, especially when the distance between patterns is measured on a correlation basis. Here we present an algorithm for the estimation of background envelope. The algorithm selects a set of concave kernels of the total profile and subsequently performs a Fourier smoothing of the trace linking these kernels. A fraction of this smoothed trace is then the background estimate. The method is illustrated for the analysis of total protein extracts of bacterial strains.
Characterisation of a glycoprotein in oviductal fluid by two‐dimensional electrophoresis and lectin binding to protein gel blots
Authors: Sutton Rosemary , Wallace Alan L. C. , Nancarrow Colin D.
Published: May 2005

Two‐dimensional electrophoresis and Western blotting with lectins were used to provide information about a glycoprotein without requiring its previous purification. The oestrus‐associated glycoprotein from ovine oviductal fluid has an isoelectric point of 4.7 and a subunit molecular weight (Mr) range of 70 000–90 000. Under non‐denaturing conditions, it is present as a high molecular weight aggregate or polymer although at low pH some monomer is also detected. The surface carbohydrate on this protein includes fucose, galactose and N‐acetyl galactosamine residues but not glucose, mannose or sialic acid.
A high resolution, rapid procedure for alpha 1‐antitrypsin phenotyping
Authors: Budowle Bruce , Murch Randall S.
Published: May 2005

An ultrathin‐layer polyacrylamide gel isoelectric focusing method is described for typing alpha 1‐antritrypsin. This method, which uses Pharmalyte pH 4.2–4.9 and a 9.5 cm or 7.0 cm inter‐electrode wick distance, clearly resolves the M1, M2, M3, S, Z and a number of rare variant allelic products. In addition, a rapid screening method for the detection of the Z, the most deficient variant, using a gel with an electrode wick distance of 5.0 cm, is also efficaceous in the detection of this and other rare allelic products.
Mechanical stabilization of large pore acrylamide gels by addition of linear polyacrylamide
Authors: Mills Nathaniel C. , Ilan Judith
Published: May 2005

Linear polyacrylamide has been used to increase the tensile strength and stability of low percentage acrylamide gels in the range of 2.5 to 6.0 % w/v acrylamide. This added strength allows handling and staining of the gel without the excessive difficulty which is normally incurred when working in this range. Addition of up to 0.6 % w/v of linear polyacrylamide gives no pore size reduction. While addition of linear polyacrylamide to acrylamide gels using bisacrylamide as a crosslinker has a hazy appearance, the use of diallyltartardiamide as a crosslinker gives the desired clarity without loss of large pore size and improves adherence to glass walls. A further advantage of acrylamide gel matrices is that the traditional methods of silver staining can be used for detection of DNA. Thus, adding linear polyacrylamide to an acrylamide gel using diallytartardiamide as a crosslinker allows easy use of low percentage acrylamide gels for separation and detection of DNA fragments in the range of 70 to 2500 base pairs.
Improved esterase D typing on ultrathin isoelectric focusing gels using narrow interval Pharmalyte carrier ampholytes (pH 4.5–5.4) in conjunction with a separator and a thickness modified pH gradient
Authors: Gill Peter
Published: May 2005

Three common alleles are found in esterase D: EsD 1, EsD 2 and EsD 5. In addition, a number of rare variants are found. The isoeiectric points of the EsD 1 and EsD 2 isoenzymes differ by only 0.02 pH units, which could lead to possible confusion between the two using conventional isoelectric focusing (IEF). Therefore, in order to ensure adequate separation of these two bands, it is necessary to use a narrow pH gradient. The separation of esterase D (EsD) has been improved on electrofocusing gels by incorporating a narrow range Pharmalyte (pH 4.5–5.4) in conjunction with the separator N‐(2‐hydroxyethyl)piperazine‐N′‐2‐ethanesulphonic acid (HEPES). This mixture resulted in the formation of a narrow pH range of pH 4.85–5.35 (the major typing bands of EsD fall within the range of pI 4.9–5.2). Resolution was further improved by the use of thickness modified pH gradients in ultrathin gels which flattened the pH gradient in the range of pH 5.0–5.1. The techniques used were shown to be effective for typing bloodstains up to 1 month old.
Sodium dodecyl sulfate agarose gel electrophoresis: Stacking of the samples in a short polyacrylamide layer
Authors: Rhalem Abdelkébir , Pery Pierre
Published: May 2005

A metod is described for obtaining a sample‐stacking effect before sodium dodecyl sulfate‐agarose gel electrophoresis. The method requires no novel equipment and uses a discontinuous polyacrylamide‐agarose horizontal slab gel system. With the buffers selected, a complex mixture of proteins can be separated, according to the molecular weight of their subunits, in 15 % w/w agarose gels. The new gel system is versatile and may offer advantages over sodium dodecyl sulfate ‐ polyacrylamide gel electrophoresis in such applications as protein elution or enzyme renaturation.
An immunochemical method for detecting an inactive enzyme (bisphosphoglyceromutase) after Cellogel electrophoresis
Authors: Calvin Marie‐Claude , Préhu Marie‐Odette , Kechemir Dalila , Villeval Jean‐Luc , Rosa Raymonde
Published: May 2005

A new method is described for detecting inactive enzymes or other proteins after Cellogel electrophoresis. The proteins were blotted from the Cellogel strip to a nitro‐cellulose membrane and then the blot was blocked before incubation in a specific antibody solution. The antigen‐antibody complex was located by a second antibody staining procedure employing peroxidase‐linked goat anti‐rabbit IgG. The method described is sensitive and could be of general use for the detection of proteins after Cellogel electrophoresis.