bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of semi‐dry and conventional tank‐buffer electrotransfer of proteins from polyacrylamide gels to nitrocellulose membranes
Authors: Tovey Euan R. , Baldo Brian A. 
Published: May 2005

AbstractThe rate of transfer of radiolabelled proteins from sodium dodecyl sulphate‐polyacrylamide gels to nitrocellulose membranes by semi‐dry blotting and electro‐transfer in a tank of buffer with and without sodium, dodecyl sulphate was quantitated. The semi‐dry blot method transferred about two thirds of most proteins in the first hour; less than one tenth of the total protein in the gel was transferred in the following hour. Conventional transfer in a tank of buffer without sodium dodecyl sulphate was dependent on the molecular weight and isoelectric point of the proteins. The efficient migration of high molecular weight and basic proteins required the presence of sodium dodecyl sulphate. The efficiency of transfer to the nitrocellulose depended both on the transfer method and on the pore size of the nitrocellulose. Almost all protein which left the gel could be accounted for when 0.1 μm pore size was used, but when 0.45 μm pore size was used in combination with semi‐dry blotting or transfer in a tank of buffer containing 0.1 % sodium dodecyl sulphate, less than 60 % of the protein could be accounted for after transfer. When transfers by two of the methods were compared using a mite extract as an antigen source and a group of miteallergic sera for detection, the same proteins were detected in each case but those which had been transferred by the semi‐dry blot method were more easily detected by the antisera used.
The application of ligand overlay of protein blots to the study of the nicotinic acetylcholine receptor
Authors: Gershoni Jonathan M. 
Published: May 2005

AbstractProtein blotting is used to identify and characterize the ligand binding site of the nicotinic acetylcholine receptor. The affinity of the complex formed on the filter can be determined by quantitative blot analyses. The precise binding‐sequence can be pinpointed by toxin overlay of proteolysed receptor α‐subunit. These techniques are now being employed in the analysis of bacterially expressed toxin binding sites.
Quantitative gel electrophoresis of polystyrene particles with 20–60 nm radii on 30% crosslinked polyacrylamide gel
Authors: Orbán László , Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractThe hypothesis was tested that polyacrylamide gel which is presumably of constant fiber structure within a practical range of gel concentrations would yield a linear Ferguson plot in the gel electrophoresis of particles in the size range of 10–100 nm. Polystyrene particles ranging from 22 to 60 nm in radius were subjected to gel electrophoresis on 30% N,N′‐methylenebisacrylamide‐crosslinked polyacrylamide (2.0 to 3.2% gel concentration) containing 50 mM 3‐[(3‐cholamidopropyl)‐dimethyl ammonio]‐1‐propane sulfonate (CHAPS). Visualization of the zones required transparent gels which were formed by polymerization both at 50 °C and in the presence of 6 M urea. Ferguson plots [log(mobility) vs. gel concentration] were progressively curved with increasing particle size from 22.4 to 59.4 nm radius. As in agarose gel electrophoresis, this curvature comprises one or several sigmoidal curve elements, presumably indicative of particle compression [1, 2]. However, by contrast with agarose gel electrophoresis, the curves can be aligned along a linear, rather than a convex axis, and may therefore be designated as “overall‐linear” in essential agreement with the hypothesis underlying this study. However, contrary to expectation, the polyacrylamide gel electrophoresis of particles in the size range of interest did not provide a practical method since the gel concentration range comprising Ferguson plots of the polystyrene particles in polyacrylamide is excessively narrow and the concentration range needed for extrapolation of the Ferguson plot to obtain free mobility values is excessively wide.
A convex “Ferguson plot” of polystyrene particles in electrophoresis on 0.25 to 2.0 % polyacrylamide (30 % Bis‐crosslinked)
Authors: Orbán László , Tietz Dietmar , Chrambach Andreas 
Published: May 2005

AbstractThe plot of log(mobility) vs. gel concentration for polystyrene particles of 26.9 and 44.2 nm radius in 30% Bis‐crosslinked polyacrylamide below 2.2 %T, containing 22.5% Metrizamide and 6 M urea, is convex. The convex plots of the two particles are essentially parallel. This parallelism suggests a retardation by a mechanism other than sieving. The parallel plots cover a range of polyacrylamide concentrations characterized by progressive fluidity as %T is lowered below 2 %T. The convex curvature renders suspect the previous free mobility and valence determinations in polyacrylamide gel electrophoresis obtained by linear extrapolation of Ferguson plots to 0 %T. Nonlinear extrapolation to 0 % T of plots of log(mobility) vs. gel concentration in the concentration range below 2 %T of 30% Bis‐crosslinked polyacrylamide for the two polystyrene particles, corrected for the effects on mobilities of Metrizamide, urea and a lower ionic strength, provides free mobility values similar to those obtained under otherwise identical conditions on agarose gel electrophoresis.
Affinity chromatography of reticulocyte lysates on haptoglobin improves two‐dimensional mapping of translation products
Authors: Rademacher Bruce E. , Steele William J. 
Published: May 2005

AbstractA simple method is described for enhancing the resolution and detectability of translation products in two‐dimensional mapping of reticulocyte lysate translation systems. Assays are fractionated by affinity chromatography on human haptoglobin‐Affi‐Gel 15 to remove hemoglobin and translation products are eluted with sodium dodecyl sulfate, buffered at pH 9.2. Translation products purified in this, way have less than 10 % of the hemoglobin present in the original assay. The recovery of translation products is about 95 % after haptoglobin chromatography and about 75 % after concentration for mapping. Hemoglobin removal not only enhances the resolution and detectability of low and moderate abundance translation products but also reduces horizontal and vertical streaking in two‐dimensional mapping without significantly altering the distribution of translation products.
A2HS*11: A new allele of alpha‐2‐HS‐glycoprotein found in Afro‐Caribbeans
Authors: Westwood Sara A. , Seaman Philip J. , Ablett Peter J. , Yuasa Isao , Weidinger Sebastian , Umetsu Kazuo 
Published: May 2005

AbstractA novel and unique protein pattern for alpha‐2‐HS‐glycoprotein (A2HS) was found in the sera from two unrelated Afro‐Caribbean donors after isoelectric focusing followed by immunofixation. In the native state the variant bands were located between A2HS 7 and A2HS 10 bands. The desialized samples showed that a cathodal variant band was indistinguishable from the A2HS 5 band. These findings indicated that both these donors possessed a variant phenotype of A2HS determined by a new allele A2HS*11, in combination with the common A2HS*1.
Analysis of two‐dimensional electrophoretic protein patterns using a video camera and a computer. II. Adaptation of automatic spot detection to visual evaluation
Authors: Prehm Joachim , Jungblut Peter , Klose Joachim 
Published: May 2005

AbstractThree two‐dimensional electrophoretic protein patterns (fluorograms) showing a representative spectrum of different qualities of resolution (spots well separated, spots embedded in “streaks” and spots of low contrast to the background) were selected to ascertain the optimum of the parameters of a computer program used for spot detection with a video camera/computer system. A “visual norm‐pattern” was constructed on the basis of the results obtained by visual evaluation of the three patterns by several test persons. The visual norm‐pattern (a graphic spot pattern) consisted of two classes of spots: “visible spots” (spots detected by all test persons) and “invisible but true spots” (spots detected by all persons, but only if the exposure time of the X‐ray film was increased). This pattern was used as a standard pattern for automatic evaluation. The automatic evaluation of the three test patterns was performed under various conditions: In the computer program two parameters critical for spot detection, parameter P1 (responsible for the separation of the spots) and P2 (responsible for the size of the spots), were varied over a broad range. In this way a third class of spots, “invisible, artifactual spots”, was revealed. The three spot classes could not be distinguished by means of the automatic system. Therefore, the visual norm‐pattern was used to adjust parameters P1 and P2 at an optimum level of sensitivity. The optimum values found for P1 and P2 allowed us to detect all the visible spots of a two‐dimensional protein pattern, and, in addition, a portion of the invisible but true spots, while none of the artifactual spots was included in the computer‐produced patterns. The P‐values chosen could be used for two‐dimensional protein patterns of different qualities. Slight deviations of the P‐values from the optimum were acceptable.
Rat liver cytosolic protein changes after ethanol exposure studied by two‐dimensional electrophoresis
Authors: Wirth Peter J. , Vesterberg Olof 
Published: May 2005

AbstractRats were fed liquid food containing ethanol in concentrations ranging from 1–5 % for 13 weeks. Livers were removed for histopathology and the liver cytosolic protein fraction was prepared and used for two‐dimensional gel electrophoresis (2D‐PAGE). Polypeptides were visualized by silver staining. Scanning was made for estimation of the relative abundance of protein in each polypeptide spot in the gels and for comparison between rats. Visual inspection and scanning of gels with the stained polypeptide spots obtained after equilibrium isoelectric focusing and non‐equilibrium pH gradient electrophoresis revealed that: (1) within the control rat and ethanol‐treated rat livers the numbers of polypeptide spots detected using isoelectric focusing in the first dimension were approximately 500 and for non‐equilibrium pH gradient electrophoresis 400; (2) in the control group the variation in the estimated amount of protein in each spot was remarkably small; (3) pronounced differences in the relative abundance of protein in several of the spots was observed in the ethanol‐exposed rats as compared to controls. Dose‐response relations and possible causes for the effects of ethanol are discussed.
Different patterns of human serum transferrin on isoelectric focusing using synthetic carrier ampholytes or immobilized pH gradients
Authors: D'Alessandro Anna M. , D'Andrea Gabriele , Oratore Arduino 
Published: May 2005

AbstractIsoelectric focusing (IEF) of human serum transferrin allows splitting of the protein pattern into three forms corresponding to the diferric, monoferric and apoform. A detailed analysis of this pattern, performed on transferrin at different degrees of iron saturation, demonstrated that free Ampholine carrier ampholytes (CA) alter the expected results, always giving a complex pattern with multiple bands. In particular, the monoferric form appears to be the predominant one, regardless of the starting saturation of transferrin. In contrast to IEF‐CA, the new technique of IEF in immobilized pH gradients (IPG), shows a much simpler pattern with the same samples. Moreover, the different transferrin forms are focused at the same pI values as in IEF‐CA but the pattern appears to correspond to the expected distribution. IPG analysis gives a pattern similar to IEF‐CA when free Ampholine CA are added either to the samples and/or as electrode solutions. A chelating action of Ampholine CA on Fe+3 might be responsible for these effects, while Immobilines, due to their different chemical nature or to the different focusing procedure, are not able to interact with iron.
Production of monospecific antisera by immunization with precipitin line leads to misinterpretation of immunoblot
Authors: Aoues Abdelkader , Huault Claude 
Published: May 2005

AbstractImmunoblotting displays an artifact in the preparation of monospecific antibodies from immunocomplexes cut out of line immunoelectrophoresis. Agarose induces the formation of antibodies towards a 68 K Da protein which is contained in commercial agarose.
Comparison of oligoclonal immunoglobulin G bands in multiple sclerosis cerebrospinal fluid by immunoblotting and immunofixation
Authors: Mehta Pankaj D. , Patrick Bruce A. , Black Jeff 
Published: May 2005

AbstractWe previously analyzed unconcentrated cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS) and other neurologic diseases by isoelectric focusing in agarose gel. We have now developed an immunoblot method for detection of oligoclonal IgG bands in unconcentrated MS CSF. The oligoclonal IgG band patterns seen after immunoblotting were compared with those of conventional immunofixation. Although immunoblotting was found to be rapid the resolution and intensity of oligoclonal IgG bands were somewhat better after immunofixation. Since immunofixation is simpler than immunoblotting, we recommend that clinical laboratories use immunofixation after isoelectric focusing to detect oligoclonal IgG bands in CSF.
Discontinuous buffer systems optimized for the agarose gel electrophoresis of subcellular particles
Authors: Orbán László , Hahn Elisabeth , Chrambach Andreas 
Published: May 2005

AbstractDiscontinuous buffer systems operative between pH 5.7 and 7.4, 0 °C, were generated, which are characterized by more rapidly displaced moving boundaries than applied previously. These allow one to resolve subcellular particles relatively rapidly and at relatively low agarose gel concentrations. A commercial mixture of DNA restriction fragments pre‐stained with ethidium bromide was found to be a suitable tracking dye for these boundaries.
Thin‐Layer agarose isoelectric focusing of alkaline phosphatase isoenzymes from human neutrophils
Authors: Brisson‐Lougarre Andrée , Vergnes Henri , Grozdea Jean , Biermé Robert 
Published: May 2005

AbstractAn improved method for thin‐layer agarose isoelectric focusing of alkaline phosphatases (AP) from human neutrophils is described. The solubilization of AP isoenzymes was studied with four detergents. The best results were obtained after sonication with Zwittergent 3–12 (1 % final concentration), followed by butanol extraction and ultracentrifugation 105 000 × g for 1 h. The cytosol can be stored at O °C or −80 °C, but not at −20 °C. Dialysis of the cytosol against a Tris buffer, pH 7.5, was imperative prior to focusing for removal of the detergent. Enzyme visualization is enhanced by incorporation of Zn Cl2 (3 mM) into the agarose gel. The focusing patterns consist of two sets of isoenzymes: two main zones with pI 6.4 and 6.8 and minor components with pI 4.2, 4.8 and 5.2.
A comparative study of protein synthesis by keratinocytes and fibroblasts in vitro using two‐dimensional gel electrophoresis and dual isotope autoradiography
Authors: Easty David J. , Patel Ketan , Dover Robin , Evans David J. , Dunn Michael J. 
Published: May 2005

AbstractA procedure is described for the detection of keratinocyte specific proteins. Fibroblasts and keratinocytes were isolated from human skin and radiolabelled in vitro. Samples were separated by two‐dimensional polyacrylamide gel electrophoresis to compare the proteins synthesised by the different types of cultured skin cells. Dual label autoradiography of samples radiolabelled with [35S]methionine and [75Se]selenomethionine was used to identify keratinocyte specific proteins. We report 45 keratinocyte‐specific components and identify some of these proteins. The differential expression of these proteins and their relevance to epidermal differentiation are discussed.
Conditions for improved adsorption of calmodulin to nitrocellulose: Detection by 45Ca binding
Authors: Hincke Maxwell T. 
Published: May 2005

AbstractThe 45Ca filtration method which allows calcium‐binding proteins to be detected after their adsorption to nitrocellulose filters was used to test conditions for improved Western blotting of calmodulin. Our results indicate that dodecyl sulphate is not efficiently removed from calmodulin during electroelution in standard transfer buffers. This detergent disrupts binding of calmodulin to nitrocellulose. Electrotransfer in potassium buffer which precipitates dodecyl sulphate leads to a 6‐fold increase in calmoculin retention by nitrocellulose. With this and other modifications, the detection limit of the 45Ca overlay method of Maruyama et al. (J. Biochem. 1984, 95, 511–519) for calmodulin is increased to 0.2 μg per lane.
Genetic variations of human serum β2‐glycoprotein I demonstrated by isoelectric focusing
Authors: Richter Antoinette , Cleve Hartwig 
Published: May 2005

AbstractHuman serum β2‐glycoprotein I was analyzed by isoelectric focusing followed by im‐munoprinting or immunoblotting with monospecific antiserum. Isoelectric focusing revealed a heterogeneous pattern consisting of 4 major and 4–5 minor bands with isoelectric points of the major bands between pH 5.4 and 6.2. Comparative analysis of sera from more than 400 healthy blood donors showed individual variations of band patterns: six different phenotypes were observed. A family study of 44 families with a total of 129 children demonstrated the genetic control of this variation. Presumably, three alleles, called B2G*1, B2G*2, and B2G*3, determine six phenotypes: B2G 1, 2, 3, 1–2, 1–3 and 2–3. The phenotype 3, however, has not been found in this study. An additional phenotype, noted in one serum specimen, was tentatively classified as B2G 2–4. The distribution of phenotypes and alleles in two populations, from Munich and from Tyrol, has been examined and the frequencies are presented. This genetic polymorphism appears not to be associated with inherited quantitative variations of β2‐glycoprotein I found earlier. The inherited variations can still be recognized after treatment of sera with neuraminidase and with endoglycosidase F, although the banding pattern is altered and shifted towards the cathode. The genetic polymorphism can, therefore, not be ascribed to variations residing in the carbohydrate side chains.
Subtyping of group specific component (GC) in microbloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection
Authors: Pflug Werner 
Published: May 2005

AbstractA powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme‐immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood microstains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.
The adsorption of large proteins in electrofocusing on immobilized pH gradients: I. Protein specificity and dependence on Immobiline and carrier ampholyte concentrations
Authors: Fawcett John S. , Chrambach Andreas 
Published: May 2005

AbstractPhycoerythrin, ferritin, urease, β‐galactosidase and thyroglobulin, with molecular masses in excess of 200 kDa, adsorb and consequently fail to migrate to, and focus at, their pI positions in electrofocusing in immobilized pH gradients at a total Immobiline concentration of 20 mM while they do focus normally in pH gradients formed by carrier ampholytes. The addition of carrier ampholytes (pH range 3.5–9.5) at concentrations of 0.1 to 5 % to the Immobiline‐containing gels reduces adsorption (desorbs) some but not all of the 5 proteins at specific Immobiline concentrations. The adsorption is not due to water redistribution and consequent reduction in gel porosity; nor is it due to conductivity minima across the pH gradient. The hypothesis that the presence of oligomeric Immobiline contributed to the protein adsorption is the subject of the accompanying report.
Fast and sensitive protein staining with colloidal acid Violet 17 following isoelectric focusing in carrier ampholyte generated and immobilized pH gradients
Authors: Patestos Nikos P. , Fauth Markus , Radola Bertold J. 
Published: May 2005

AbstractA new method is described for fast and sensitive staining of proteins following isoelectric focusing in carrier ampholyte and immobilized pH gradient polyacrylamide gels. After fixation with trichloroacetic acid the gels are stained for 5–10 mm with 0.1–0.2 % colloidal Serva Violet 17 (generic name: Acid Violet 17; Color Index No. 42 650) in 10 % w/v phosphoric acid. After staining for only 0.5–3 min, major zones, corresponding to 100–500 ng protein, are visible without destaining on a weak background. Detection of minor components requires destaining with 3 % w/v phosphoric acid for 5–80 min depending on gel thickness (120–500 μm) and type of support (fabric reinforcedversusgels backed to a polyester film). For selected pH marker proteins (bovine serum albumin, carbonic anhydrase, horse myoglobin) a staining sensitivity of 1–2 ng/mm2protein is found. Dye elution from stained fabric reinforced gels with 50 % v/v dioxane‐water, followed by absorbance measurements results in a linear relationship over a range of 1–100 μg marker proteins. Staining colloidal Serva Violet 17 is the only method available for fast and high sensitivity and low background staining of immobilized pH gradient gels, without interference from selective dye binding in different pH ranges. Staining with the colloidal dye is convenient by avoiding organic solvents with unpleasant vapors and potentially hazadous.
Two‐dimensional electrophoresis. Analysis of tubulin proteins and peptides in neuronal and non‐neuronal tissues using immobilized pH gradients
Authors: Field Deborah J. , Lee James C. 
Published: May 2005

AbstractThe accurate identification of the individual protein products of multi‐gene families is essential to the interpretation of data from a wide range of experimental approaches including molecular biology, protein chemistry, and cell biology. We have adapted immobilized pH gradient isoelectric focusing to provide high resolution of tubulin proteins [1, 2]. Here we use these techniques to investigate the heterogeneity of tubulin in several neuronal and non‐neuronal tissues to provide an accurate evaluation of isotubulin composition. Of the ten sources examined, the greatest number of isotubulins was found in whole adult brain. Tubulin isolated from either neonate human or rat brain consists predominantly of the more basic alpha and beta isotubulins found in adult brain. Cerebrum, cerebellum, medulla and caudate nucleus all contain the same large number of isotubulins as in whole brain, but in varying proportions. Liver, kidney and spleen isotubulin populations are all similar to each other and consist of a simpler distribution than any neuronal tissue examined. The majority of the tubulin protein in these non‐neuronal tissues is composed of only the most basic alpha tubulins and intermediately‐charged beta tubulins. No isotubulins were identified that were unique to these three non‐neuronal tissues. Tubulin from neuroblastoma cells has an isotubulin distribution grossly similar to non‐neuronal sources but additionally contains two basic beta isotubulins found in adult brain that are absent from non‐neuronal tissues.