bluuebunny/crossref_metadata_2025_split huggingface dataset:
Native immunoblotting: Transfer of membrane proteins in the presence of non‐ionic detergent
Authors: Bjerrum Ole J. , Selmer Johan C. , Lihme Allan 
Published: May 2005

AbstractFor analysis with monoclonal antibodies against discontinuous epitopes and for performance of functional characterization it is important to maintain proteins native during electrophoresis and electroblotting. However, in vitro membrane proteins only remain soluble and native in the presence of non‐ionic detergent, which, on the other hand blocks the subsequent protein immobilization on nitrocellulose. With model proteins from human erythrocytes, platelets and‐serum, it has been examined if electroblotting to nitrocellulose can be performed from agarose gels in the presence of non‐ionic detergent (native blotting). Each of the following procedures were successful: (i) employment of detergents with low critical micellar concentration; e. g. Berol EMU‐043 (C 16–18, E10); (ii) insertion of a 2–3 mm thick detergent‐free agarose layer between the gel and nitrocellulose; (iii) transfer at pH 11.8 of immunoprecipitated proteins after gel wash; (iv) binding via covalent coupling to vinylsulfone activated nitrocellulose. The procedure to be chosen depends on the type of analysis and on the required binding efficiency. Compared to immunoblotting after sodium dodecyl sulfate‐gel electrophoresis, native immunoblotting was 10–30 times more sensitive. Of general applicability is the easy transfer from crossed immunoelectrophoresis gels and the possibility for performance of covalent coupling to nitrocellulose during transfer with preservation of the original binding properties of the matrix.
The use of colloidal metal particles in protein blotting
Authors: Moeremans Marc , Daneels Guy , De Raeymaeker Marc , De Wever Bart , De Mey Jan 
Published: May 2005

AbstractTwo procedures are described for staining the overall protein pattern on transfer membranes. Both methods involve the use of colloidal metal particles. AuroDye staining is based on the interaction of anionic colloidal gold particles with cationic proteins at low pH. The method is highly sensitive, but limited to nitrocellulose and polyvinylidene difluoride membranes. FerriDye comprises an incubation in a cationic iron (hydrous) oxide sol and subsequent intensification with potassium hexacyanoferrate in acidic medium. FerriDye is of intermediate sensitivity, but there are no limitations concerning membranes. It can be used on nitrocellulose‐, nylon‐and polyvinylidene difluoride‐based membranes. An immunodetection procedure is described which uses colloidal gold‐labelled secondary antibodies to visualize antigen‐antibody interactions. This signal can be significantly enhanced by silver. The procedure explained uses a new light‐insensitive and user‐friendly silver enhancement system.
Glycosyltransferases as probes for non‐reducing terminal monosaccharide residues on nitrocellulose immobilized glycoproteins: The β (1–4) galactosyltransferase model
Authors: Parchment Ralph E. , Shaper Joel H. 
Published: May 2005

AbstractThis paper reviews a technique for detecting non‐reducing terminal N‐acetyl‐glucosamine residues on glycoproteins in a mixture of proteins resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immobilized on nitrocellulose by electrophoretic transfer. Detection is accomplished by fluorography after enzymatic modification with galactosyltransferase (UDP‐galactose: N‐acetyl‐D‐glucosaminyl‐glycopeptide 4‐β‐D‐galactosyltransferase, E.C. 2.4.1.38), which transfers radiolabeled galactose from its nucleotide sugar substrate, UDP‐[6‐3H]‐galactose, into any immobilized glycoprotein with a non‐reducing terminal 2‐acetamido‐2‐deoxy‐D‐glucose (GlcNAc) acceptor site. The kinetics of radiolabeling are independent of the amount of immobilized glycoprotein substrate. Half‐maximal incorporation of [6‐3H]galactose occurs by 5 h and saturation by 16 h. We have used enzyme blotting with galactosyltransferase: (i) to detect non‐reducing terminal GlcNAc residues on isolated glycoproteins, (ii) to detect, in combination with endo‐β‐N‐acetylglucosaminidase F digestion, Asn‐linked oligosaccharides on glycoproteins, and (iii) to identify a subset of glycoproteins with non‐reducing terminal N‐acetylglucosamine residues in both F9 and PYS murine teratocarcinoma cell extracts, two of which appear to be unique to PYS cells. Because the family of glycosyltransferases recognizes a large variety of non‐reducing terminal monosaccharide residues, this technique, termed enzyme blotting, could be applied to the analysis of any non‐reducing terminal monosaccharide. This method could be used to obtain initial information about the carbohydrate structures of a particular glycoprotein without any prior purification steps. This technique may also be potentially useful in elucidating the functional contribution of non‐reducing terminal monosaccharide residues in intercellular recognition and adhesion.
Protein antigen purification by preparative protein blotting
Authors: Montelaro Ronald C. 
Published: May 2005

AbstractRecent reports have described procedures for recovering proteins from nitrocellulose membrane blots, either by dissolution of the membrane with acetone or dimethyl‐sulfoxide (DMSO) or by specific desorption of the immobilized proteins with acetonitrile or pyridine. In the current review, various procedures have been evaluated for the recovery of protein antigens from blots on nitrocellulose, Zeta Probe nylon, Immobilon, or glass fiber membranes. The results of this survey indicate that elution with appropriate concentrations of acetonitrile affects equally efficient desorption of proteins bound to nitrocellulose, Zeta Probe, or Immobilon membranes, while formic acid was effective in eluting proteins from glass fiber or Zeta Probe membranes. Although nitrocellulose blots could be dissolved in acetone or DMSO for antigen production, other membrane matrices were resistant to dissolution by organic solvents, except for Immobilon in DMSO. The use of volatile solvents permits the reconstitution of the isolated lyophilized protein into the buffer of choice with retained protein structure or biological activities (e. g. antigenicity). Nitrocellulose appeared to be most versatile for preparative protein blotting because it displays a protein binding capacity equivalent to other membranes, effectively retains a variety of large and small molecular weight proteins, and is compatible with reversible protein staining by Ponceau S.
Characterisation of allergens by protein blotting
Authors: Tovey Euan R. , Baldo Brian A. 
Published: May 2005

AbstractTechniques developed for protein blotting have been applied to the characterisation of allergen extracts. Such extracts, used for the diagnosis and therapy of allergic diseases, are highly variable in their content of active components. Protein blotting allows the direct identification of those components in an extract which bind human immunoglobulin E and cause allergic symptoms. It also allows the components to be defined in terms of molecular weight, isoelectric point and the frequency and intensity of IgE‐binding by individual allergens. Because of the large number of samples which can be handled in a single experiment, protein blotting of allergens allows the identification of the individual allergen recognition spectra of large numbers of allergic patients. The resolution of components, loss of antigenicity, and aspects of blocking and probing which are important in identifying IgE‐binding components are reviewed. A table of allergen sources investigated by protein blotting is included and important sources of allergens, such as pollens, mites and fungi are discussed in detail.
Detection by polyacrylamide gel electrophoresis of the human serum amyloid A protein in inflammatory sera
Authors: Peristeris Platon , Peyret Michel , Bernon Hélène , Bienvenu Jacques , Beal Brigitte , Laurent Philippe 
Published: May 2005

AbstractHuman serum amyloid A protein in inflammatory sera is easily identified by simple protein staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis as the lowest molecular weight detected proteic band. Western blotting followed by immunoblot has been used to demonstrate this result. By contrast, serum amyloid A protein is not detectable by these combined techniques in normal sera.
Experimental and theoretical investigation of the stability of stepwise pH gradients in continuous flow electrophoresis
Authors: Kuhn Reinhard , Wagner Horst , Mosher Richard A. , Thormann Wolfgang 
Published: May 2005

AbstractIsoelectric focusing in the continuous flow mode can be more quickly and economically performed by admitting a stepwise pH gradient composed of simple buffers instead of uniform mixtures of synthetic carrier ampholytes. The time‐consuming formation of the pH gradient by the electric field is thereby omitted. The stability of a three‐step system with arginine – morpholinoethanesulfonic acid/glycylglycine – aspartic acid is analyzed theoretically by one‐dimensional computer simulation as well as experimentally at various flow rates in a continuous flow apparatus. Excellent agreement between experimental and theoretical data was obtained. This metastable configuration was found to be suitable for focusing of proteins under continuous flow conditions. The influence of various combinations of electrolytes and membranes between electrophoresis chamber and electrode compartments is also discussed.
Determination of transferrin isoproteins in human cerebrospinal fluid using isoelectric focusing and zone immunoelectrophoresis assay
Authors: Petrén Sven 
Published: May 2005

AbstractThe relative amount of a transferrin isoprotein with isoelectric point 5.7 (Tf5.7) and its ratio to total transferrin concentration (Tfratio) in blood serum and plasma has earlier been shown to be a good marker for alcohol abuse and, recently, after occupational exposure to organic solvents. To facilitate study of the relatively low concentration of the transferrin forms in cerebrospinal fluid, the procedure for iron saturation and an earlier method using isoelectric focusing separation in gel followed by quantification with zone immunoelectrophoresis assay were improved. The detection limit was lowered more than tenfold, allowing determination of total transferrin amount in un‐concentrated cerebrospinal fluid. Samples from ten healthy volunteers were examined. The Tfratio in cerebrospinal fluid was found to be higher than in blood.
Two‐dimensional electrophoresis of cellular proteins of human cultured fibroblasts: Search for sex‐chromosome‐dependent variants
Authors: Klösser‐Ziegelmayer Sabine , Cleve Hartwig 
Published: May 2005

AbstractCellular proteins of 30 cultivated human fibroblast cell lines were analyzed by two‐dimensional gel electrophoresis (2‐DE) with special regard to sex‐chromosome‐dependent differences. The cell lines were obtained from 28 individuals; these included seven normal females (46, XX), eight normal males (46, XY) and thirteen patients with numerical aberrations of their sex chromosomes. Cellular proteins were labelled metabolically with [3H] leucine. Unfractionated cell proteins were prepared as well as a Triton‐extractable and a structural protein fraction. The 2‐DE patterns showed some individual variations: proteins designated as fp 45, fp 39 and fp 29–28, respectively, differed in their position. The protein fp 27 was either present or absent in the 2‐DE patterns. The spots of the proteins fp 63 and fp 20 showed marked variations in intensities. Fp 20 was somewhat stronger in 2‐DE fluorograms from cell lines with more than two X chromosomes (47, XXX; 48, XXXX; 49, XXXXY). The other variations were not related to the sex chromosomes. We did not find protein variations dependent on the presence or absence of the Y chromosome or dependent on the presence of one or two X chromosomes. The various gonosomal aberrations were not characterized by specific changes of their 2‐DE patterns.
Differences in protein staining by Coomassie Brilliant Blue and neutron activated Coomassie Brilliant Blue dyes
Authors: Gersten Douglas M. , Wolf Pamela H. , Zapolski Edward J. 
Published: May 2005

AbstractThe degree of interaction between protein and stains depends in large part upon the protein's content of basic amino acids. We have compared “staining” characteristics of Coomassie Brilliant Blue R‐250 with a dye that was rendered radioactive by neutron bombardment. The protein‐dye intensities were compared to amino acid composition as in our previous study (Electrophoresis 1986, 7, 327–332). While the data for “colored” dye reflected involvement of basic amino acid residues, quite unexpectedly, the radioactive dye appeared to act in part with the acidic amino acids. This may be due to enhanced binding of dye impurities or radiolysis products.
A rapid screening technique for identifying murine monoclonal antibodies to human apolipoprotein A‐I isoforms by Western blot analysis
Authors: Henderson L. Omar , Graiser Samuel R. , Phillips Donald J. , Gomo Zvenyika A. R. , Aloisio Carol H. 
Published: May 2005

AbstractA rapid method for determining the reactivity of murine monoclonal antibodies with apolipoprotein A‐I (Apo A‐I) isoforms has been developed. The method consists of a rapid (1–2 h) prescreening of fusion well culture fluids by using an automated immunofluorescent assay (IF A) to identify fusion well cultures producing monoclonal antibody against Apo A‐I and high‐density lipoprotein (HDL). Immediately following the IFA procedure, immunodetection of the reactivity pattern of selected fusion well culture fluids is determined by using transblotted isoforms of Apo A‐I previously separated by isoelectric focusing. Strips are prepared and stored at 4 °C in protein containing blocking solution before use. Preselected culture fluids could be tested within 6 h for qualitative reactivity with Apo A‐I isoforms. Using this procedure we selected several monoclonal antibody‐producing cultures for further studies on HDL metabolism based on their differing reactivities with Apo A‐I isoforms. This technique should be easily transferable to other laboratories for assessing proteins having multiple isoforms since the crucial steps of separation and blotting can be optimized and performed before actual testing for antibody reactivity.
Improved rehydration by controlled drying of polyacrylamide gels for hybrid isoelectric focusing
Authors: Rossmann Ute , Altland Klaus 
Published: May 2005

AbstractA procedure is described to improve the properties of dried gels, containing immobilized pH gradients, for rehydration in the presence of urea. Triton X‐100, reducing agents and carrier ampholytes. When gels are dried by a fan in ambient atmosphere for the shortest time interval required to achieve an evenly flat surface, rehydration in the presence of 8 mol/L urea and 30 or 5 mL/L of Triton X‐100 is completed within 4 or 1 h, respectively. These rehydration properties can be maintained by storing the dried and sealed gels at −20 °C.
Fast horizontal electrophoresis. II. Development of fast automated staining procedures using PhastSystem™
Authors: Olsson Ingmar , Wheeler Richard , Johansson Catherine , Ekström Björn , Stafström Nils , Bikhabhai Rama , Jacobson Gunilla 
Published: May 2005

AbstractThe development of equipment for fast automated staining is described. It is possible to handle staining procedures with up to 20 steps and nine different solutions. To increase the reaction rate in the reaction chamber, the gels are rotated and high temperatures are used. The temperature in the reaction chamber is controlled between room temperature and 50 °C. Increased temperature, above 20 °C, generally results in faster staining and destaining. However, some reactions proceed better at a low temperature, including fixation of proteins with TCA, and the development step in silver staining, where increased temperatures cause a high background stain. Silver in silver staining, where increased temperatures cause a high background stain. Silver staining using acidic silver nitrate solution is preferred, due to easy preparation and good storage stability of the reagents. This method also causes little precipitation of silver on the walls of the reaction chamber. Silver staining is accomplished within one hour. Staining with PhastGel Blue is accomplished within 30 min.
An improved voltage measurement device for gel electrophoresis in tube apparatus
Authors: Orbán László , Fawcett John S. , Sullivan James V. , Chidakel Burt E. , Chrambach Andreas 
Published: May 2005

AbstractA device for the measurement of voltage across tube gels was designed and constructed which allows one to measure voltage during electrophoresis without any manipulation of the gel electrophoresis apparatus or gel tube and with the elimination of a source of inaccuracy in previous such devices.
Approach to stationary two‐dimensional pattern: Influence of focusing time and Immobiline/carrier ampholytes concentrations
Authors: Görg Angelika , Postel Wilhelm , Günther Siegfried , Weser Johann , Strahler John R. , Hanash Samir M. , Somerlot Luke , Kuick Rork 
Published: May 2005

AbstractHorizontal two‐dimensional (2‐D) electrophoresis with immobilized pH gradients (IPG) in the first dimension for buffer soluble proteins and for complex proteins solubilized in the presence of Nonidet P‐40 (Görg et al., Electrophoresis 1987, 8, 45–51), has been extended to analyze basic proteins of yeast cells focused under non‐equilibrium and equilibrium conditions. Transient state isoelectric focusing (IEF) in IPG gels revealed sample smearing and background staining, displaying horizontal streaks in the resultant 2‐D patterns. Inclusion of 0.5 % carrier ampholytes (CA) to the IPG gel (IPG‐CA), resulted in the formation of many sharp protein bands after transient state IEF with resultant distinct spots in the 2‐D patterns; however, resolution was poor and the gel contained heavy background staining. With prolonged focusing time, background staining disappeared and there was less difference in the final steady state IEF patterns obtained with IPG and IPG‐CA. Reduction of the Immobiline concentration to one third the manufacturer's recommended amount did not improve IEF resolution with respect to streaking and background staining under either transient state or equilibrium conditions. In general, spot intensities were less on 2‐D gels using diluted IPG gels than with “standard” IPG gels. Optimization of 2‐D electrophoresis with IPGs in the first dimension was strongly related to IEF conditions. The use of IPG gels focused to equilibrium should not only improve inter‐gel reproducibility and resolution but also the quality of the final 2‐D patterns with respect to background staining and horizontal streaking.
Increased sensitivity for Coomassie staining of sodium dodecyl sulfate‐polyacrylamide gels using PhastSystem Development Unit
Authors: Heukeshoven Jochen , Dernick Rudolf 
Published: May 2005

AbstractStaining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate‐polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.
Cardiac muscle protein analysis by high‐resolution and microscale two‐dimensional gel electrophoresis
Authors: Tanaka Takao , Kawamura Keishiro 
Published: May 2005

AbstractAn improved method of two‐dimensional gel electrophoresis of small muscle samples is described. Isoelectric focusing of cardiac whole muscle homogenate in agarose gels containing urea and detergent has a markedly increased resolution. Equilibration of the first‐dimensional gels with detergent before application to the second‐dimensional gels is unnecessary in this system. By applying this method to rat cardiac whole muscle, high‐molecular weight proteins, such as myosin heavy chains, are focused on the first‐dimensional gels and, in addition, minor components are resolved on the second‐dimensional gels, without loss during equilibration with detergent. The two‐dimensional electrophoretic patterns of rat cardiac whole muscle obtained with this method reveal numerous clearly separated spots. By analyzing the two‐dimensional electrophoretic patterns of rat cardiac whole muscle and various rat cardiac fractions, and by staining the calcium‐binding proteins with “Stains‐all”, we identified some cardiac muscle components, such as myosin heavy and light chains, actin, tropomyosin, and troponin C, but additional work is required to identify the remaining spots. The two‐dimensional electrophoretic system described here makes possible the effective resolution of whole cardiac muscle homogenate from small samples, and looks promising as an aid to muscle research.
Low voltage isoelectric focusing for the study of red cell acid phosphatase polymorphism: Improved phenotyping and better agreement with the Hardy Weinberg distribution
Authors: Destro‐Bisol Giovanni , Ranalletta Dalila 
Published: May 2005

AbstractA low voltage isoelectric focusing (IEF) method for typing human red cell acid phosphatase (ACPl) polymorphism is reported. A clear differentiation of the ACPl*A, ACPl*B and ACPl*C allelic products is obtained by minimizing diffusion of the bands during electrophoresis and during enzyme visualization by using a hydrophilic cellophane film soaked with the substrate solution, heated to 50 °C, and by reading without incubation in a moist chamber. A population study of ACPl, carried out with this method, on a sample of 624 unrelated individuals of the population of Rome (gene frequencies: ACPl*A = 0.267; ACPl*B = 0.680; ACPl*C = 0.053), showed a good agreement between the observed and expected phenotype distribution (χ = 1.35; 0.70 > p > 0.50).
Isoelectric focusing of catalase from acatalasemic mouse and human blood, and cultured human skin fibroblasts
Authors: Ogata Masana , Satoh Yukinori 
Published: May 2005

AbstractHemolysates of normal, heterozygous hypocatalasemic and acatalasemic mice and of Japanese acatalasemic subjects were separated into three fractions, A, B and C, by DEAE‐cellulose column chromatography, and pI values of A, B and C fractions were determined by isoelectric focusing. The pI value of catalase in the A, B and C fractions increased in the order of normal, hypocatalasemic and acatalasemic mouse blood. The results obtained from Japanese acatalasemic blood samples showed that the pI values of catalase in the A, B and C fractions were similar to those in normal blood. Catalase in Japanese acatalasemic cultured skin fibroblasts was also analyzed by isoelectric focusing. The pI values of catalase in the extract from the cultured skin acatalasemic fibroblasts was similar to that in normal fibroblasts.
An improved method of high‐voltage isoelectric focusing on cellulose acetate membranes
Authors: Toda Tosifusa , Sano‐Shiba Kiyoko , Cho Hiroko , Soon Poh Lin , Nakao Makoto , Ohashi Mochihiko 
Published: May 2005

AbstractAn apparatus and the procedure for cellulose acetate isoelectric focusing were refined and optimized for routine use in clinical laboratories. The space in the electrophoretic chamber was minimized to maintain high humidity during the run. The water content of the thin cellulose acetate membranes in the chamber was well controlled during isoelectric focusing and the effect of atmospheric carbon dioxide was also excluded. The temperature of the membranes was kept below 2 °C, even under conditions of high electric field strength for faster isoelectric focusing of high‐molecular‐weight proteins. No special training was required for technicians in clinical laboratories to obtain reproducible isoelectric profiles of human serum proteins.