bluuebunny/crossref_metadata_2025 huggingface dataset:
The effect of prestaining prior to immunoreaction during electrophoretic blotting of proteins
Authors: Tracy Russell P. , Monkovic Don , Andrianorivo Aurelie , Calore James , Mackay Alastair
Published: May 2005

We have used six different monoclonal and polyclonal antibodies, four different antigen preparations and two different detection systems to compare Western blotting with Coomassie Blue prestained gels (Jackson and Thompson, Electrophoresis, 1984, 5, 35–42) to blotting of unstained gels with Amido Black or Fast Green post‐transfer staining of the nitrocellulose. Contrary to a recent report (Harper et al., Anal. Biochem., 1986, 157, 270–274), in which post‐staining with Coomassie Blue was determined to severely inhibit immunoreactivity, we find using prestained Coomassie Blue gels to be extremely useful in most cases, although, rarely, inhibition of the immune reaction may take place. Post‐staining with Amido Black or Fast Green may also prove useful, but is not recommended since similar inhibition occurs, and there are the added disadvantages of not being able to “pre‐view” the gel pattern and not being able to use stored gels. Further studies indicated that the rare loss of immunoreactivity seen with Coomassie Blue prestained gels is most likely due to the necessary fixation step, and not the stain itself (as has been suggested), since the protein‐dye complex is dissociated during transfer.
Simplified molding procedure for ultrathin‐layer polyacrylamide slab gels using methacrylate supports and unsilanized glass plates: Hybrid isoelectric focusing on ultrathin immobilized pH gradients
Authors: Pascali Vincenzo L. , Destro‐Bisol Giovanni , d'Aloja Ernesto
Published: May 2005

A simplified procedure for the preparation of ultrathin polyacrylamide slab gels is reported. The molding cells consist of an unsilanized glass plate and methacrylate plate or acryl‐coated glass plate. Owing to the repelling properties of acrylate the ultrathin gels spontaneously detach from the acrylate surface on unfastening the molds, firmly adhering to the unsilanized glass. Gels as thin as 0.20–0.25 mm could be polymerized without resorting to silanized glass plates or polyester films. The procedure is especially suitable for sticky gels, such as highly cross‐linked polyacrylamide gels and gels with immobilized pH gradients.
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

For 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

Two 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

This 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

Recent 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

Techniques 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

Human 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

Isoelectric 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

The 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

Cellular 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

The 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

A 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

A 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

The 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

A 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

Horizontal 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

Staining 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

An 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

A 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

Hemolysates 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

An 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.
A quantitative theory of the stern electric double layer
Authors: Michov Budin M.
Published: May 2005

A quantitative theory of the Stern electric double layer is suggested. It is based on the view that every ion possesses a geometrical and an electrokinetic radius, that the ionic atmosphere begins from the geometrical one, and that the difference between these radii is the Stern quantity Δ. The equations of the mentioned radii and the quantity Δ are established and the values of the different potentials characterizing an ion and its ionic atmosphere are determined.
Enhancing the sensitivity of DNA detection and recovery from agarose gels
Authors: Georges Fawzy , Girard Sophie , Chibbar Ravindra N. , Constabel Friedrich
Published: May 2005

By inserting nitrocellulose strips into agarose gels alongside the electrophoresed lanes and passing an electric current perpendicularly in the direction of the strips, highly efficient transfer of DNA bands onto the membrane in the form of concentrated dots is achieved. DNA detection limits by this technique are enhanced, at least three times as visualized by ethidium bromide fluorescence and at least twice more by radiolabeling.
A motor‐driven syringe‐type gradient maker for forming immobilized pH gradient gels
Authors: Fawcett John S. , Sullivan James V. , Chidakel Burt E. , Chrambach Andreas
Published: May 2005

A motor driven gradient maker based on the commercial model (Jule Inc., Trumbull, CT) was designed for immobilized pH gradient gels to provide small volumes, rapid stirring and delivery, strict volume and temperature control and air exclusion. The device was constructed and by a convenient procedure yields highly reproducible gradients either in solution or on polyacrylamide gels.
Improved transfer of high molecular weight proteins using fabric‐reinforced soft polyacrylamide gels
Authors: Starita‐Geribaldi Mireille , Giudicelli Jean , Sudaka Pierre
Published: May 2005

Rapid transfer of electrophoretically separated, high molecular weight proteins from fabric‐reinforced, soft, low‐concentration polyacrylamide gels (3.5 %T, 2.6 %C) is described. Polyester fabrics proved useful for gel reinforcement during vertical slab gel electrophoresis. The fabric‐reinforced gel retains its dimension during transfer, equilibration and drying, facilitating comparison of protein bands in the separation gel and transfer matrix.
Application of Neuhoff's optimized Coomassie Brilliant Blue G‐250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films
Authors: Peisker Klaus
Published: May 2005

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G‐250 in phosphoric acid/ammonium sulfate, was applied to ultrathin‐layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfatepolyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel‐Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.
Linear Ferguson plots of polystyrene sulfate size standards for the quantitative agarose gel electrophoresis of subcellular particles
Authors: Hahn Elisabeth , Wurts Linda , Tietz Dietmar , Chrambach Andreas
Published: May 2005

Accurately standardized commercial polystyrene sulfate particles in agarose gel electrophoresis yield linear Ferguson plots at pH 7.4 over a gel concentration range up to 0.9% agarose which do not exhibit any significant sigmoidal curve elements, using either a discontinuous buffer system or a continuous buffer. Ferguson plots of these standard‐sized particles were evaluated using alternatively a linear or convex model, by means of a newly developed set of programs (to be used in conjunction with program M‐LAB) which (i) is sufficiently user‐friendly to allow for quantitative agarose gel electrophoresis of subcellular‐sized spherical particles based on their convex Ferguson plots with the same operational simplicity previously available for linear Ferguson plots only; (ii) simultaneously and interactively analyzes the Ferguson plots of all particles under consideration on the basis of an extended Ogston model.
Comparison between isoelectric focusing methods for the detection of orosomucoid phenotypes
Authors: Montiel Maria Dolores , Carracedo Angel , Lopez‐Rodriguez Isidro , Rodriguez‐Calvo Maria Sol , Concheiro Luis , Huguet Emilio , Gené Manuel
Published: May 2005

Orosomucoid (ORM) polymorphism was investigated by different methods including isoelectric focusing in acid pH ranges followed by silver staining, print immunofixation of desialyzed ORM, fixation using a lectin from the sea‐weed Codium tomentosum, isoelectric focusing followed by immunofixation in miniaturized gels and isoelectric focusing in immobilized pH gradients. Population genetics studies were carried out in Galicia (NW Spain) and two new ORM variants were found.
Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level
Authors: Orbán László , Chrambach Andreas
Published: May 2005

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single‐stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.
Peptide mapping of polypeptides separated by two‐dimensional electrophoresis: Protease digestion directly on the two‐dimensional gel followed by electrophoresis in reverse direction
Authors: Zivy Michel , Granier Fabienne
Published: May 2005

A method is described which allows to reveal simultaneously the proteolytic patterns of numerous polypeptides separated by two‐dimensional electrophoresis. After two‐dimensional electrophoresis, the gels were dipped successively in buffers for preequilibration, protease digestion, and reequilibration. They were then returned to the electrophoresis tank, and electrophoresis was continued for a short time. After silver staining, digestion products appeared, lined up behind the original polypeptide spots. The method allows proteolytic patterns of numerous polypeptides to be viasualized simply and quickly. Among proteins of wheat leaves, 31 groups of related polypeptides were found according to the similarity of their proteolytic patterns.
A simple method for preparation of molecular weight marker proteins for sodium dodecyl sulfate‐polyacrylamide gel electrophoresis by photopolymerizations of hemoglobin subunits
Authors: Sato Hiroshi , Aono Sachiko , Semba Reiji , Kashiwamata Shigeo
Published: May 2005

Photoirradiation of globin, obtained from human hemoglobin, in the presence of oxygen and protoporphyrin produced a series of polymers of globin subunits ranging from dimer to dodecamer. These polymers are useful as molecular weight markers because they cover a relatively wide range of molecular weights (Mr 15 500–186 000) with a constant and narrow interval of Mr 15 500.
Specific antigen detection by immunoelectroendosmosis
Authors: Lewis W. H. P. , Sun K. K. Y.
Published: May 2005

A novel approach to the immunological stage of immunoblotting is described. The method employs the phenomenon of electroendosmosis to bring the antibodies sequentially into contact with the antigen. Using a three‐stage peroxidase antiperoxidase system for human group specific protein (Gc) phenotyping, it has been shown that the method has comparable sensitivity, is more rapid and is more economical of antibodies than a similar multi‐stage immerse‐and‐wash system.
Two‐dimensional electrophoresis of human lymphocyte proteins: Two‐dimensional polymorphisms and paternity testing
Authors: Waldinger Dorothea , Cleve Hartwig
Published: May 2005

Genetic polymorphisms of seven human lymphocyte proteins, analyzed by two‐dimensional electrophoresis, were evaluated in respect to their suitability for paternity testing. Current data of an enlarged family and population study for five proteins (p23, p30, p40, p60, p66), already described for a smaller population sample of Southern Germany, are presented together with evidence for a new polymorphic protein (p42), recently observed in our survey. These six proteins occurred in isoelectric focusing as two different variants, acidic (a) and basic (b). The genetic basis of the protein variations was ascertained (i) by the presence of homozygous and heterozygous phenotypes, (ii) by the Mendelian mode of transmission of the variants as allelic gene products within 17 families and (iii) by the demonstration of a gene‐dosage dependence comparing the spot intensities in homozygous and heterozygous phenotypes. For quantitative data, laser densitometric scanning of the protein spots followed by computer‐assisted quantitative evaluation of the spot intensities was performed. The allele frequencies of the polymorphic proteins were calculated from the phenotype distributions within a sample of 56 unrelated individuals from Southern Germany. Gene frequencies of the common alleles ranged between 0.991 and 0.518. To discuss the suitability of the two‐dimensional polymorphisms for paternity testing the theoretical exclusion probabilities were assessed for seven polymorphic proteins observed in our population sample, the six polymorphisms with two alleles described here and a further polymorphism (p75) with six alleles. For five proteins (p23, p40, p42, p66 and p75) we found sufficiently high values for the theoretical exclusion probabilities, ranging from 10% to 34%. For the seven polymorphic proteins, which could be analyzed on a single two‐dimensional gel, we calculated a combined theoretical exclusion probability of 67%.
Forensic aspects of haptoglobin: Electrophoretic patterns of haptoglobin allotype products and an evaluation of typing procedure
Authors: Teige Brita , Olaisen Bjørnar , Pedersen Lilian , Teisberg Per
Published: May 2005

A procedure used for haptoglobin (Hp) typing in paternity cases has been evaluated. All serum samples have been subtyped with a one‐dimensional isoelectric focusing/immunoblotting method, and samples with rare or questionable patterns have been further examined by two‐dimensional electrophoresis with isoelectric focusing in the first dimension followed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the second dimension. The electrophoretic Hp‐patterns of common and rare α‐ and β‐chain variants are shown, including allotype patterns of two new β‐chain variants and three new α‐chain variants. Retyping of nearly 2000 individuals at intervals between 1 to 12 months revealed a typing error frequency of about 0.3 %, which is considered acceptable, provided new blood samples are required in every case of paternity exclusion. Comparison of typing results obtained with the present procedure and with routine starch gel electrophoresis in more than 5000 serum samples gave conflicting results in 6 samples. The sensivity of the described one‐dimensional subtyping method was slightly better than that of starch gel electrophoresis. In 4110 unrelated individuals, involved in cases of disputed paternity the Hp 2SS 0.038, Hp 2FF 0.004, and Hp 3 (Johnson) 0.0005. These allele frequencies give a theoretical paternity exclusion rate of 32.5%, which is in accordance with the observed rate in 2200 paternity cases with more than 600 non‐fathers. It is concluded that the present procedure represents a definite improvement for Hp subtyping in practical paternity diagnostics. Preliminary results with retyping of weak Hp patterns using a staining technique involving the biotin/avidin complex indicate that the sensitivity of the one‐dimensional subtyping method may be substantially increased. Dried blood stains, containing approximately 1–3 μL blood, have been typed with good results employing the latter staining technique. The applicability of the one‐dimensional subtyping method in forensic casework is presently being investigated.
The group specific component/vitamin D binding protein (GC/DBP) system in the analysis of disputed paternities
Authors: Constans Jacques , Cleve Hartwig
Published: May 2005

The group‐specific component (GC) was discovered in 1959, and in the same year a vitamin D binding protein (DBP) in human plasma was found; however, their identity was established as late as 1975. In the GC/DBP system three common alleles, GC*1F, GC*1S, and GC*2, determine six GC phenotypes: 1F, 1S, 2, 1F‐1S, 2‐1F and 2‐1S, these common alleles having been found in all human populations studied. In addition, more than 120 GC variants have been discovered, with varying frequencies in different populations. The distribution of the common GC phenotypes and the presence of rare GC variant phenotypes render the GC/DBP system useful for the analysis of disputed paternities.
Identification of group specific component/vitamin D‐binding protein (GC/DBP) mutants by isoelectric focusing in immobilized pH gradients
Authors: Constans Jacques , Cleve Hartwig
Published: May 2005

The six common genetic types of the group specific component/vitamin D‐binding protein (GC/DBP) system are usually classified by isoelectric focusing in carrier ampholytes, followed by visualization of the GC proteins by immunoprinting with monospecific antiserum. In addition, more than 120 mutant GC types have been discovered. For their identification additional methods were necessary, including poly acrylamide gel electrophoresis, isoelectric focusing in the presence of 3 M urea as well as isoelectric focusing in immobilized pH gradients. The application of the last method is described in detail and several examples of GC/DBP mutants identified thereby are presented.
Acid phosphatase typing for breeding nematode‐resistant tomatoes by isoelectric focusing with an ultranarrow immobilized pH gradient
Authors: Günther Siegfried , Postel Wilhelm , Wiering Henk , Görg Angelika
Published: May 2005

The genetic variants of tomato acid phosphatase (Aps‐1) systems have been analyzed by isoelectric focusing in immobilized pH gradients (IPG). By using an ultranarrow pH 4.25–4.55 IPG gel, the two genotypes Aps‐11 and Aps‐1+, differentiating tomato variants into nematode‐resistant or nematode‐susceptible plants, are separated into two sharp zones over a distance of 2.5 cm with isoelectric points of 4.37 and 4.43, respectively. Under these conditions, silver staining of the Aps‐1 variants proved to be superior to enzyme staining. By applying more than 50 samples on one IPG gel, this method proved to be a powerful tool for reliable tomato nematode resistance screening.
Electric field gradients and band sharpening in DNA gel electrophoresis
Authors: Slater Gary W. , Noolandi Jaan
Published: May 2005

A mathematical study of the effect of non‐uniform electric fields on the width of DNA electrophoretic bands is presented. Using a simple model, we show that field gradients sharpen these bands during an experiment if the corresponding gradient of electrophoretic velocity is large enough. This is in agreement with experimental results indicating that narrower bands form when pulsed field electrophoresis is carried out in the presence of field gradients. Moreover, it is shown that there is in fact an optimal experimental duration that maximizes separation. Finally, gradients are also predicted to reduce the relative mobilities of the DNA fragments, which is a serious drawback of this technique.
Density gradient electrophoresis of cells in a reversible gel
Authors: Plank Lindsay D. , Kunze M. Elaine , Gaines Richard A. , Todd Paul
Published: May 2005

Density gradient electrophoresis permits the separation of cell types according to surface charge density with high resolution. Any source of flow compromises the resolving power of density gradient electrophoresis. Although procedures have been devised to successfully counteract electroosmotic and convective flows, the final collection of separands requires that they be pumped out of the electrophoresis column. Experiments were therefore designed to test the hypothesis that this flow could also be eliminated by trapping the separated bands in a gel, from which they could be collected by slicing the gel cylinder. Glutaraldehyde‐fixed rat and rabbit erythrocytes were used as test particles in a phosphate‐buffered isotonic Ficoll‐sucrose density gradient in a 2.2 cm diameter, thermostated vertical glass column that could be opened at both ends. Two types of agarose were used as gel polymers: Electrophoresis grade agarose (J. T. Baker Chemical Co.) at final concentrations of 0.1 to 0.25 % and SeaPrep ultralow gelling agarose (Marine Colloids Div., FMC Corp.) at a final concentration of 1.0 %. Electrophoretic separability of the test particles and fluid stability were tested independently at 55 °C and 32 °C at which the two agaroses were, respectively, liquid. The experiments demonstrated that the higher temperatures required and the presence of agarose compromised neither the stability of the density gradient nor the migration properties of the cells, and cells can be separated in a sol at a temperature that is compatible with cell viability.
Blotting from PhastGel media after horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
Authors: Jägersten Christina , Edström Annica , Olsson Birgitta , Jacobson Gunilla
Published: May 2005

An improved procedure, “thermoblotting”, is described for transferring proteins by diffusion from PhastGel™ Gradient media to an immobilizing matrix after horizontal sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After electrophoresis the gels were left on the separation bed of PhastSystem™, the blotting matrix was applied and a transfer temperature was selected between 5–70 °C. An experimental series at fixed diffusion times showed that the transfer yield was significantly increased with temperature. The evaluation was done visually after staining of the blots with colloidal gold. An evaluation study comparing nitrocellulose, nylon, and polyvinyl‐idenedifluoride of different pore sizes is also reported. Finally, the transfer efficiencies for 125I‐labelled bovine serum albumin and soybean trypsin inhibitor were estimated using four different blotting procedures: two diffusion blotting techniques and two electrophoretic blotting techniques (tank vs. semi‐dry).
An evaluation of two‐dimensional electrophoresis for detection of human serum proteins in acute myocardial infarction
Authors: Marshall Thomas , Williams John , Williams Katherine M.
Published: May 2005

High resolution two‐dimensional electrophoresis indicates that serum proteins previously only detected in patients with acute myocardial infarction (AMI) (Gomo et al., Electrophoresis 1983, 4, 298–302) are also present in the serum protein patterns of other patients (AMI negative or demonstrating acute phase response) and are faintly detected even in controls. Thus, these proteins are not specific to AMI and are probably acute phase reactants. However, they do demonstrate a characteristic time course response in sequential samples from AMI patients.
Two‐dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): Method, reproducibility and genetic aspects
Authors: Görg Angelika , Postel Wilhelm , Domscheit Albert , Günther Siegfried
Published: May 2005

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two‐dimensional electrophoresis with immobilized pH gradients (IPG 4–7 and IPG 6–10) in the first dimension. Highly reproducible two‐dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety‐specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.
The use of two‐dimensional gel electrophoresis in the analysis of organ‐specific maize proteins
Authors: Goday Adela , Torrent Margarita , Ludevid M. Dolors , Puigdomènech Pere
Published: May 2005

Two‐dimensional gel electrophoresis with non‐equilibrium pH gradient electrophoresis in the first dimension and sodium dodecyl sulfate‐polyacrylamide gels in the second dimension has been used for the analysis of organ‐specific proteins in maize. The method has been used to study the whole protein pattern of developing organs and adult leaves as well as protein patterns of in vitro translation. Examples of two‐dimensional immunoblotting and in vitro translation of endosperm‐specific proteins are also shown. Two‐dimensional gel electrophoresis appears as an essential analytical step in the identification of organ‐specific proteins and for the detection of the protein products related to organ‐specific genes identified by other means.