bluuebunny/crossref_metadata_2025_split huggingface dataset:
Electrophoretic migration velocity of amphiphilic proteins increases with decreasing Triton X‐100 concentration: A new characteristic for their identification
Authors: Børsum Tone , Bjerrum Ole J. 
Published: May 2005

AbstractThe relation between the electrophoretic migration velocity and the concentration of the non‐ionic detergent Triton X‐100 in the buffer was studied by crossed immunoelectrophoresis of hydrophilic and amphiphilic proteins. The migration velocity of hydrophilic human plasma proteins was not affected by the presence of Triton X‐100 in the agarose gels in the concentration range of 1–0 % v/v. In contrast, amphiphilic proteins, like the human erythrocyte proteins glycophorin, band 3 protein and acetylcholinesterase, and the plasma high density lipoprotein (HDL) apolipoprotein, showed increasing migration velocity with decreasing detergent concentration in the gels. An increase of 20–80 % was observed for different amphiphilic proteins when the Triton X‐100 concentration was lowered from 1 % to 0.03 % v/v. This probably reflects the influence of the size of the bound detergent micelle on the charge density of the protein. Thus, by performing corssed immunoelectrophoresis with different concentrations of Triton X‐100 in the first‐dimensional gel it is possible to identify amphiphilic proteins. Presence of 1 % v/v Triton X‐100 in the second‐dimensional gel ensures reliable identification of the precipitates.
Electrophoresis on uncrosslinked polyacrylamide: Molecular sieving and its potential applications
Authors: Tietz Dietmar , Gottlieb Melvin H. , Fawcett John S. , Chrambach Andreas 
Published: May 2005

AbstractUncrosslinked polyacrylamide, a viscous liquid, provides a molecular sieving effect in electrophoresis qualitatively the same as that of crosslinked gels. This finding confirms previous results by others using uncrosslinked polyacrylamide mixed with various gel matrices. It provides a sieving medium constituted by an entirely and necessarily random association of inert fibers, as stipulated for interpretation of migration by the Ogston theory. Ferguson plots of several proteins in uncrosslinked polyacrylamide exhibit Yo values smaller and less divergent from one another than in 5 % crosslinked gels, suggesting a curvature of the Ferguson plots of crosslinked polyacrylamide gels at low %T. If linearity of these plots at low %T, 0 %C could be demonstrated, a means would be found to determine accurate values of free mobility by their linear extrapolation to 0 gel concentration. Potentially, uncrosslinked polyacrylamide at low concentrations may provide a tool for the analytical sieving of subcellular particles. However, electrophoresis in uncrosslinked polyacrylamide at concentrations of less than 10 % is perturbed by convection, and thus these potential applications will depend on combining it with a suitable anticonvective device.
A device for preparative elution electrophoresis using a polyacrylamide gel slab
Authors: Carpenter Helen C. , Skerritt John H. , Wrigley Colin W. , Margolis Joel 
Published: May 2005

AbstractA compact device, capable of being clipped onto the end of a 3 mm thick analytical polyacrylamide slab gel, has been developed for the collection of resolved protein zones as they elute from the end of the gel. Its unique feature is that eluted proteins were collected between two polyacrylamide‐embedded paper membranes and removed either by continuous or intermittent buffer elution. Because of this direct coupling to large gels identical to those used for analytical electrophoresis, protein zones were obtained with high resolution. The method was applicable to non‐denaturing cathodic and anodic electrophoresis, as well as sodium dodecyl sulfate electrophoresis. Medium‐scale quantities (up to 10 mg/cm2 cross‐sectional area) of protein were fractionated in as little as 4 h with high recoveries.
Fast silver staining of polyacrylamide gels at elevated temperatures
Authors: Biel Hans J. , Gronski Peter , Seiler Fritz R. 
Published: May 2005

AbstractRenouncing protein fixation with aldehydes, the polyacrylamide gels are incubated in alcoholic solutions of silver nitrate containing acetic acid at 60–70 °C. for a short time and developed with NaOH, also at 60–70 °C. Using various methods of protein fixation, the detection limits of human albumin were determined for 0.75 and 1.5 mm thick polyacrylamide gels with appropriate thickness correction. An overlay technique with silver nitrate containing polyacrylamide gels is described. Dispersing Coomassie Brilliant Blue G‐250 in a solution containing HNO3 and NH4NO3 allows rapid silver staining of prestained gels.
Mathematical simulation of isotachophoresis boundary between protein and weak acid
Authors: Shimao Kazuo 
Published: May 2005

AbstractMathematical simulation of an isotachophoretic boundary between protein and weak acid was made on the assumption of steady state. In order to deal with a protein molecule, a Gaussian model for charge distribution of protein molecule was constructed, based on the assumption that acidic and basic residues on a protein molecule dissociate independently of each other. The results were roughly in agreement with protein stacking experiments with regard to boundary thickness and concentration of the stacked zone.
Electrophoretic characterisation of human parotid saliva protein fractions isolated by preparative isoelectric focusing
Authors: Hallinan Frank M. , Rose Marie , Eagleton Marie , Tempany Edward 
Published: May 2005

AbstractHuman parotid saliva proteins fractionated by preparative isoelectric focusing into 3 main fractions have been characterised electrophoretically and immunochemically. The anodic proteins constitute a polymorphic family of collagenase and trypsin sensitive, immunochemically related, phosphoproteins. Many of these contain a common polypeptide of Mr 23 000 either alone or in combination with other lower (Mr 21 000) or higher (Mr 25 000) molecular weight polypeptides. Parotid amylase was substantially purified by this technique and an antiserum against these fractions reacted monospecifically with amylase in a number of systems. The cathodic proteins are all collagenase sensitive proteins with Mr values of 12 500 to 77 000.
Comparison between agarose gel electrophoresis (Panagel) of cerebrospinal fluid with silver staining, and polyacrylamide disc gel electrophoresis for demonstration of the oligoclonal pattern in neurological disorders
Authors: Plan Elisabeth , Seneterre Jean‐Bernard , Caudie Christiane , Quincy Claude 
Published: May 2005

AbstractA high resolution agarose gel system (Panagel) with silver staining and polyacrylamide disc gel electrophoresis were used to demonstrate oligoclonal bands in cerebrospinal fluid (CSF) from 150 patients identified as having either inflammatory neurological diseases (110 patients) or non‐inflammatory neurological diseases (40 patients). The detection of oligoclonal band patterns in high resolution agarose electrophoresis is sensitive: 65% (polyacrylamide gel electrophoresis: 50%) and specific: 95% (polyacrylamide gel electrophoresis: 73%) with respect to inflammatory neurological diseases. This procedure is easier to perform than polyacrylamide disc gel electrophoresis and is recommended for routine examination of CSF especially for the detection of oligoclonal bands.
Involvement of carbohydrate moieties in the heterogeneities of human blood clotting factor IX
Authors: Polack Benoǐt , Freyssinet Jean‐Marie 
Published: May 2005

AbstractPurified human blood clotting factor IX, although homogeneous by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, showed two peaks when analyzed by crossed immunoelectrophoresis in the presence of calcium. Differential affinity of lectin from wheat germ (WGA) for the two forms of factor IX, detected by crossed affino‐immunoelectrophoresis, demonstrated that carbohydrate moieties are involved in factor IX heterogeneities. The slow migrating peak has a higher affinity for WGA (Kd= 5.26 × 10−7 M) than the fast migrating peak (Kd= 1.29 × 10−5 M). This study emphasizes the usefulness of lectins as tools to assess homogeneity and integrity of glycoproteins.
On‐line fiber optic UV detection cell and conductivity cell for capillary zone electrophoresis
Authors: Foret František , Deml Mirko , Kahle Vladislay , Boček Petr 
Published: May 2005

AbstractSimple detection cells for on‐line UV absorbance and conductivity detection in capillary zone electrophoresis are described. The UV detection system employs a commercial photometer and optical fibers which are in direct contact with the outer walls of the separation capillary and conduct the light beam perpendicularly across the migrating zones. The conductivity detection cell consists of platinum wires protruding the wall of the separation capillary and thus in direct contact with the electrolytes inside. The electronic system is adopted from a commercially available conductivity detector for isotachophoresis.
Isoelectric spectrum and titration curves of Aleutian disease virus proteins
Authors: Kierek‐Jaszczuk Danuta , Kaaden Oskar‐Rüger 
Published: May 2005

AbstractThree major virus‐specific proteins (Mr = 85 000, 75 000 and 25 000) were detected in purified preparations of the cell culture‐adapted Aleutian disease virus (ADV) SL3 isolate according to their molecular weight, isoelectric point and titration curve, For all structural proteins, amino acid sequence homologies were demonstrated by proteolytic peptide mapping. The virus‐induced protein Mr 75 000, detected only in lysates of infected cells, revealed a different proteolytic peptide map. It was shown that each of the structural proteins, although demonstrated to be homogeneous in molecular weight, consists of multiple species differing in their isoelectric points. Protein modifications are discussed as possible cause for the charge variations of ADV structural proteins.
Advantages of enzyme‐immuno‐assay after blotting in bloodstain grouping: Application to the haptoglobin groups
Authors: Hoste Bernadette 
Published: May 2005

AbstractEnzyme‐immuno‐assay after Western blotting with anti‐haptoglobin and anti‐immunoglobulin conjugated‐peroxidase or‐alkaline phosphatase increases the sensitivity of haptoglobin visualization. The detection limit in serum could be improved from 1:8 to 1:256. This method was applied to bloodstains with improved results.
Immobilized pH gradients in capillary tubes and two‐dimensional gel electrophoresis
Authors: Hochstrasser Denis , Augsburger Valérie , Funk Matthieu , Appel Ron , Pellegrini Christian , Muller Alex F. 
Published: May 2005

AbstractIsoelectric focusing in mixed carrier ampholyte‐immobilized pH gradients (CA‐IPG) is an effective way to separate proteins by charge. A method to prepare CA‐IPG in capillary tubes and the description of the equipment used are outlined. Two‐dimensional gel electrophoresis patterns of human serum and liver biopsies, with isoelectric focusing and CA‐IPG as the first dimension, are presented. The comparison with two‐dimensional gel electrophoresis patterns obtained by conventional carrier ampholyte pH gradient separation shows the excellent resolution and potential of this technique.
Separation by hybrid isoelectric focusing of normal human plasma transthyretin (prealbumin) and a variant with a methionine for valine substitution associated with familial amyloidotic polyneuropathy
Authors: Altland Klaus , Banzhoff Angelika 
Published: May 2005

AbstractThe human plasma transthyretin (TTR, prealbumin) variant TTR(Met30) has been separated from normal TTR(Val30) by double one‐dimensional electrophoresis using polyacrylamide gel electrophoresis followed by hybrid isoelectric focusing. The procedure appears to be appropriate for identification by the screening of patients with familial amyloidotic polyneuropathy of Portuguese type I. This is the second example to support the recently expressed hypothesis (Altland et al., Electrophoresis 1986, 7, 251–259) that substitutions of electrically neutral amino acids (e. g. methionine for valine at position 30) in a polypeptide chain (e. g. the TTR monomer) may be detected by isoelectric focusing under denaturing conditions (e. g. hybrid isoelectric focusing in the presence of 8 M urea, 50 mM dithiothreitol) when the exchange is close to a charged amino acid (e. g. histidine at position 31, pI (TTR) = 5.7, pKa (His) = 6.0).
Elimination of point streaking on silver stained two‐dimensional gels by addition of iodoacetamide to the equilibration buffer
Authors: Görg Angelika , Postel Wilhelm , Weser Johann , Günther Siegfried , Strahler John R. , Hanash Samir M. , Somerlot Luke 
Published: May 2005

AbstractSilver staining artifacts, unrelated to protein, such as horizontal lines and vertical micro or point streaking on two‐dimensional gels with carrier ampholytes or immobilized pH gradients in the first dimension are efficiently eliminated by adding iodoacetamide to the equilibration buffer of the first‐dimensional gel.
Isoelectric focusing‐affinity immunoblot analysis of mouse monoclonal antibodies to the four human IgG subclasses
Authors: Hamilton Robert G. , Roebber Marianne , Reimer Charles B. , Rodkey L. Scott 
Published: May 2005

AbstractIsoelectric point (pI) microheterogeneity, immunoreactivity and specificity of murine monoclonal anti‐human IgG subclass antisera (MoAb) were evaluated in parallel by isoelectric focusing (IEF)‐affinity immunoblotting and enzyme‐linked immunosorbent assay (ELISA). Ascites protein bands in the pH 3 to 9 region of Coomassie Blue‐stained IEF‐polyacrylamide gels corresponded to mouse host proteins such as albumin, transferrin and immunoglobulins. Bands in the pH 5.5 to 8.0 region were shown to be murine IgG by direct blotting onto nitrocellulose followed by detection with conjugated anti‐mouse IgG. Use of IgG myeloma (antigen)‐coated nitrocellulose in the IEF‐affinity immunoblot allowed detection of the charge micro‐heterogeneity of the MoAbs. As a group, the MoAbs had pIs ranging from 6.1 to 7.8 with a 0.1 to 0.6 pH unit spread. There were 1 to 5 major dense bands flanked by up to 4 minor fainter bands. The pI values and banding patterns as determined by IEF‐affinity immunoblot analysis were consistent both within and between blots. Semi‐quantitative estimates of binding specificity in the IEF‐affinity blot compared well with cross‐reactivity data obtained from a quantitative ELISA. IEF‐affinity immunoblotting is a useful analytical tool for defining the microheterogeneity of monoclonal antibody pI's and for monitoring antibody specificity. Both parameters are indicators of consistency of antibody produced in culture or in ascites by hybridomas which have been repeatedly frozen and thawed over several years.
A voltage gradient‐induced arrest of circular DNA during agarose gel electrophoresis
Authors: Serwer Philip , Hayes Shirley J. 
Published: May 2005

AbstractDuring the gel electrophoresis of double‐stranded DNA, interaction with the gel fractionates the DNA by molecular weight and conformation. Here, it is shown that open circular bacteriophage λ DNA (Mr 32.1 × 106) migrates into a 0.2% agarose gel during a first electrophoresis at 0.34 V/cm, but does not migrate when the voltage gradient is subsequently raised to 6.0 V/cm for a second, orthogonal electrophoresis. Linear DNA of the same molecular weight does migrate at 6.0 V/cm. The data suggest that the arrest observed at 6.0 V/cm is caused by threading of circular DNA by gel fibers. These observations have produced a procedure of two‐dimensional agarose gel electrophoresis capable of detecting comparatively small amounts of circular DNA. The concept of threading is potentially useful for understanding of DNA fractionations obtained with pulsed electrical fields.
Use of secondary antibodies for visualization of bound primary reagents in blotting procedures
Authors: Ramlau Jakob 
Published: May 2005

AbstractSecondary antibodies are used for visualization of bound primary ligands in blotting procedures in several formats. The term “secondary antibody” usually denotes an anti‐immunoglobulin from a species different from the one producing the primary antibody. This secondary antibody is modified so as to produce a visible signal by conjugation with enzyme, fluorophore, radioisotope or suitable particulatematerial. The characteristics of these antibodies will influence both sensitivity and specificity of the assays in which they are used. Thorough knowledge of these characteristics is thus needed for critical evaluation of results. This paper discusses the production and reactivity of secondary antibodies, their immunochemistry and their possible side reactions. Alternative secondary reagents are also discussed with respect to specificity and sensitivity.
Enzymoblotting: Visualization of electrophoretically separated enzymes on nitrocellulose membranes using specific substrates
Authors: Ohlsson Bertil G. , Weström Björn R. , Karlsson Börje W. 
Published: May 2005

AbstractEnzymoblotting is a technique for the detection of electrophoretically separated enzymes after transfer to nitrocellulose (NC) membranes and subsequent incubation with suitable substrates. After agarose gel electrophoresis or isoelectric focusing in ultrathin polyacrylamide gels, blotting of the separated proteins/enzymes to NC membranes was performed by capillary forces for 30 min. Hydrolases, e. g. pancreatic proteinases, were visualized using amino acid/peptide substrates based either on the β‐naphthol or para‐nitroanilide (pNA) group. Both types of substrates were used with good results, but the pNA substrates provide greater specificity and sensitivity. An oxidoreductase, lactate dehydrogenase, was visualized using an electron transfer dye staining method. Enzymoblotting also allows detection of inactive proenzymes since conversion to active enzymes in situ on the NC membrane may be performed by pre‐incubating the NC membranes with activation enzymes. The enzymoblotting technique offers several advantages over traditional gel staining methods. The enzymes are immobilized on the NC membrane, preventing diffusion during substrate incubation, and as a result, high resolution is retained even after long incubation times; the product obtained as a result of the enzyme activity can be chemically derivatized and bound to the membrane, thus widening the categories of possible substrates; subsequent incubation of the same NC membrane in various substrate solutions can be performed; enzymoblotting can be used in combination with other techniques, e. g., immunoblotting for parallel structural and functional studies.
Separation of red cell membrane proteins by urea‐Triton‐polyacrylamide gel electrophoresis in one‐ and two‐dimensional systems
Authors: Rouyer‐Fessard Philippe , Lecomte Marie‐Christine , Boivin Pierre , Beuzard Yves 
Published: May 2005

AbstractUrea‐Triton polyacrylamide gel electrophoresis (UT‐PAGE) has been used to separate, in one dimension, membrane proteins present in red blood cells and hemoglobin chains associated with cell ghosts. The membrane proteins are especially well resolved in the high molecular weight range. The protein fractions were further characterized by two‐dimensional gel electrophoresis with UT‐PAGE in the first dimension followed by sodium dodecyl sulfate (SDS)‐PAGE and phosphorylation in the presence or absence of cAMP. A good recovery of spectrin and of band III was obtained in the two‐dimensional system. The proportion of α, β, Gγ and Aγ globin chains can be determined in comparison to the membrane proteins.
A simple, inexpensive method for the salt‐free concentration of proteins for analysis by two‐dimensional gel electrophoresis
Authors: Maresh Grace A. , Dunbar Bonnie S. 
Published: May 2005

AbstractA rapid, inexpensive method for the salt‐free concentration of small quantities of proteins for analysis by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) has been developed. Proteins adsorbed to diatomaceous earth are subsequently removed using either sodium dodecyl sulfate or urea solubilization reagents for 2D‐PAGE analysis. This procedure has been found to concentrate proteins having wide ranges of molecular weight and charge. It is also valuable for the concentration of large numbers of small samples from cells cultured in vitro.
Cryoglobulinemia: Analysis of isotype, idiotype and antibody activity by composite gel electrophoresis and immunoblotting
Authors: Chu Jia‐Li , Gharavi Azzudin E. , Elkon Keith B. 
Published: May 2005

AbstractImproved methods for high resolution composite gel electrophoresis under dissociating conditions and electrophoretic transfer of immunoglobulins (Mr 150 000‐1 800 000) to nitrocellulose have been developed. Using these techniques and highly specific antisera to detect either light or heavy chains on nitrocellulose transfers, the immunoglobulin and clonal composition of washed cryoglobulins (3 μg each) could be determined within two days. The results were confirmed by isoelectric focusing and immunoblotting of selected samples. In addition, the method was used for detection of andiotypic determinant and rheumatoid factor activity within components of the cryoglobulins.
Hydrophobic interaction electrophoresis under high hydrostatic pressure: Study of the effects of pressure upon the interaction of serum albumin with a long‐chain aliphatic ligand
Authors: Masson Patrick , Reybaud Joël 
Published: May 2005

AbstractHydrophobic affinity electrophoresis under high hydrostatic pressure has been developed to study the interaction between fatty acid‐free bovine serum albumin and a long‐chain aliphatic ligand physically immobilized within the gel matrix. From apparent association constants at various pressures and temperatures, apparent thermodynamic parameters including the volume change in binding were calculated. The results are as expected for hydrophobic interactions between the long‐chain alkyl ligand and a high‐affinity long‐chain fatty acid binding site. The feasibility of high‐pressure affinity electrophoresis is demonstrated. This new high‐pressure technique provides a direct means for studying quantitatively the effects of pressure upon protein‐ligand interactions. It could become a suitable tool for the investigation of protein binding sites' topography.
Wheat cultivar identification by a totally automatic softlaser scanning densitometry and computer‐aided analysis of protein electropherograms
Authors: Autran Jean‐Claude , Abbal Philippe 
Published: May 2005

AbstractA computer‐aided analysis based on soft‐laser densitometry of electrophoretic patterns is described. The system has the potential to automatically identify wheat cultivars from normalized sodium dodecyl sulfate – polyacrylamide gel electropherograms. The principle emcompasses autocalibration of the relative mobilities of components of the unknown pattern using standards of Mr that bracket each pattern on the pictures, automatic identification of peaks and baseline correction, and simplified estimation of the peak intensities. The algorithm provides a normalized array that can be automatically compared with a library of standard cultivar patterns in order to determine those having the highest similarity percentage. The outputs of the program are (1) relative similarity plots which show the percentage of similarity between the unknown cultivar to that of each of the common standard cultivars put in a pattern library, (2) the names of cultivars in the library having the most similar patterns and (3) computer‐generated electrophoretic graphics of these cultivars. This system is not intended to identify closely related cultivars but it is particularly recommended for cultivar identification without well‐trained personnel, familiar with band location and nomenclature.