bluuebunny/crossref_metadata_2025 huggingface dataset:
Capillary zone electrophoresis and micellar electrokinetic chromatography of 4‐aminobenzonitrile carbohydrate derivatives
Authors: Schwaiger Hannes , Oefner Peter J. , Huber Christian , Grill Eduard , Bonn Günther K.
Published: May 2005

Aldoses, ketoses and uronic acids were derivatized successfully within 15 min at a temperature of 90°C by reductive amination with 4‐aminobenzonitrile. Subsequently, the derivatives were separated as their borate complexes by capillary zone electrophoresis, using 175 mM borate buffer, pH 10.5, as carrier. The electrophoretic mobilities were determined by the complex stability, which was found to depend on the number of hydroxyl groups on any given carbohydrate derivative, the presence of substituents, and most strongly on the configuration of the vicinal hydroxyl groups at C‐3 and C‐4 in aldoses and uronic acids, and with regard to ketoses on those at C‐4 and C‐5. Time of analysis could be reduced considerably by the use of micellar electrokinetic chromatography, which separated 4‐aminobenzonitrile sugar derivatives on the basis of their differential partitioning into an electroendosmotically driven aqueous phase and into sodium dodecyl sulfate micelles. Optimum resolution was achieved with a Tris‐phosphate buffer, pH 7.5, containing 100 mM of sodium dedecyl sulfate. The method made it possible to resolve several carbohydrates which had not been resolved successfully by means of capillary zone electrophoresis, such as glucose and fructose. Moreover, separation selectivity could be adjusted by varying the capillary temperature. Finally, on‐column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 1 fmol and a concentration sensitivity of 0.3 μM.
The validity of the Smoluchowski equation in electrophoretic studies of lipid membranes
Authors: Egorova Elena M.
Published: May 2005

The Helmholtz‐Smoluchowski theory, widely used for the calculation of zeta potential from the measured electrophoretic mobility (EPM), is known to be invalid in the region where the mobility is affected by the surface conductivity and polarization of the electrical double layer. In this region, the zeta potential found according to the Smoluchowski equation (ζsm) is not identical to the true electrostatic potential at the hydrodynamic plane of shear (ζ) which is considered in the Gouy‐Chapman‐Stern theory of the electrical double layer. As a result, ζsm cannot be used for the subsequent calculation of surface potential and surface charge density of a membrane studied. Here we suggest a simple way, allowing one to decide between the validity and nonvalidity of the Smoluchowski equation in various sets of experimental conditions used in electrophoretic measurements on lipid membranes. We calculated the dimensionless criterion Rel, accepted in the Dukhin theory of electrophoresis as a measure of the extent of the influence of surface conductivity and double layer polarization on EPM. The Rel changes, with membrane charge density, ionic strength and vesicle radius, were found to be helpful in finding the combinations of these three parameters, corresponding to the validity of the Smoluchowski equation.
Polypeptide cartography of Spiroplasma taiwanense
Authors: Humphery‐Smith Ian , Guyonnet Franck , Chastel Claude
Published: May 2005

Spiroplasma taiwanense is the first member of the Class Mollicutes to be subjected to polypeptide cartography using computerized image analysis. The small genome size characteristic of this group was shown to code for low numbers of polypeptides when compared to other bacterial species. Silver‐stained two‐dimensional electrophoresis gels, following separation by either isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (ISO‐DALT) or nonequilibrium pH gradient electrophoresis (NEPHGE), were used to create databases from 10 and 6 gels, respectively, for each technique and produced, respectively, 263 and 287 replicated spots. Polypeptides were mapped with respect to molecular mass and glyceraldehyde‐3‐phosphate dehydrogenase carbamylation standards. Of interest was the unexpectedly high percentage (50.2%) of the total normalised optical intensity associated with all 263 spots detected by ISO‐DALT electrophoresis, having been contributed by just 29 dominant protein spots. These 29 polypeptides are to be given priority in microsequencing and microanalysis aimed at their identification.
Application of micellar electrokinetic chromatography to the quality control of pharmaceutical formulations: The analysis of xanthine derivatives
Authors: Korman Milos , Vindevogel Johan , Sandra Pat
Published: May 2005

Micellar electrokinetic chromatography (MEKC) has been applied to the analysis of three different drugs. Although belonging to different therapeutic classes, all these drugs contain xanthine derivatives as active substances. Pen‐toxifylline was separated from eight related xanthines. Quantitative MEKC was applied to determine impurities (caffeine and xanthine) in the purified drug at the 0.05–0.1% level and also for the determination of the active substance in Agapurin® tablets. Ethofylline and theophylline were separated from ephedrine and mebrophenhydramine and determined in Xantedrylettae® tablets while caffeine was separated from mephenhydramine and determined in Kinedryl® tablets. In all cases, simple borate buffers with sodium dodecyl sulfate as the surfactant were satisfactory and little separation optimization was required. The relative standard deviation (RSD) of the migration times was better than 1% while the RSD of the observed areas was better than 2%. This demonstrates that MEKC is a valuable alternative for the traditional high‐performance liquid chromatography analysis of drugs and drug formulations.
Influence of structure on the behavior of flavonoids in capillary electrophoresis
Authors: Pietta Piergiorgio , Mauri Pierluigi , Bruno Anna , Gardana Claudio
Published: May 2005

In an attempt to elucidate the relation between structure and electrophoretic behavior, the effect of pH, surfactant and buffer on electrophoretic mobilities and resolution of several flavonoids, differing in structure, has been investigated. Ring‐B substitution and the presence of a free hydroxy group at C7 of ring A play an important role, whereas O‐methoxylation is less significant. Glycosylation also contributes in determining different degrees of complexation with borate, thus affecting the mobilities.
Gel dependence of electrophoretic mobilities of double‐stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis
Authors: Gast Frank‐Ulrich , Sänger Heinz Ludwig
Published: May 2005

Double‐stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B‐form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either ≥ 2.5% agarose or ≥ 8% acrylamide with high crosslinking (19:1 acryl‐amide: N,N′‐methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 ± 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small ≈︁ 70% base‐paired rod‐like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140–230 bp, whereas 130 ± 20 bp can be determined from electron micrographs and 123–126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low‐crosslinked polyacryl‐amide gels (2.6%C) is comparable to the determination by alternative methods.
Quantification of silver‐stained proteins resolved by two‐dimensional electrophoresis: Genetic variability as related to abundance and solubility in two maize lines
Authors: Damerval Catherine
Published: May 2005

Relative abundar ce and solubility of proteins from etiolated coleoptiles of maize were investigated using two‐dimensional electrophoresis (2‐D PAGE). Automatic quantification of silver‐stained polypeptides on replicate 2‐D gels made it possible to test the linearity of the relationship between spot integrated optical density and protein amount in the range from 15 μg to 135 μg per gel, in two inbred lines. A linear response was found for more than 60% of the spots in each genotype. When a linear response was found in both lines for a given spot, the slope values were similar in 94% of the cases, indicating the reliability of silver staining for polypeptide quantification. The parameters of the curves allowed the definition of protein classes of different abundances that could be compared for genetic variability between the two lines. From a comparison between standard 2‐D patterns (trichloroacetic acid‐acetone extracted proteins) and patterns obtained from Tris buffer extracted proteins, it appeared that 92% of the proteins visualized in the standard gels were soluble. No difference in genetic variability, ether qualitative or quantitative, was evidenced between the various classes of abundance, or between soluble and insoluble proteins.
Horizontal polyacrylamide gel electrophoresis for the separation of DNA fragments
Authors: Haas Hildegard , Budowle Bruce , Weiler Günter
Published: May 2005

A discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5–9%T), the concentration of the crosslinker piperazine diacrylamide (2–5%CPDA), as well as the concentration of formate in the gel (40–120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.
An investigation of the factors controlling the staining and destaining of electrophoresis gels
Authors: Hitchman Michael L. , Ekstrom Bjorn
Published: May 2005

A general analysis is developed for the effect of mass transport and kinetic parameters on the rate of staining and destaining of electrophoresis gels. The various contributions to these overall processes are discussed and, in particular, the ways in which the rate of solution flow, temperature and gel properties can influence staining procedures are highlighted. It is shown that for reproducible, rapid and uniform staining a rotating gel system, analogous to a rotating disc, provides the necessary controlled laminar flow. The theoretical equations derived are compared with experiments and it is shown that at high gel rotation speeds mass transport in solution does not have a major controlling influence on rates of staining and destaining. The temperature dependence of these rates also suggests that there is no significant control by the rate of interaction between stain and protein molecules. The major controlling factor under these conditions is then concluded to be the transport of stain in the gel itself, and the theoretical analysis and time‐dependent experimental results allow a determination of the corresponding diffusion parameters.
Iron‐Induced conformational change in human lactoferrin: Demonstration by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analysis of effects of iron binding to the N and C lobes of the molecule
Authors: Ying Li , He Jianglin , Furmanski Philip
Published: May 2005

Analysis of Fe‐saturated‐ and apo‐lactoferrin by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) without heating the samples prior to application revealed a substantial difference in mobility. The mobility shift was fully reversible on repetitive removal and readdition of Fe. Binding of a single Fe to the N‐lobe binding site was sufficient to cause the gel shift; binding of a second Fe to the C‐lobe site did not further alter mobility. Removal of Fe from the N lobe of Fe2 lactoferrin did not restore mobility to the position of apolactoferrin. No change in mobility with Fe binding was detected in N and C lobes isolated from intact lactoferrin by controlled trypsin digestion. The data indicate that a conformational change induced by Fe binding to a single site on lactoferrin is detectable by SDS‐PAGE and that this change requires an intact molecule, possibly due to the need for interactions between the two homologous lobes of the molecule.
Use of antibodies against synthetic peptides for analysis of molecular multiplicity: Human deoxyribonuclease I polymorphism
Authors: Yasuda Toshihiro , Nadano Daita , Takeshita Haruo , Kishi Koichiro
Published: May 2005

Eight different antibodies were raised in rabbits and chickens by injecting them with two synthetic N‐ and C‐peptides, which corresponded to the N‐ and C‐terminal 15 residues of human deoxyribonuclease I (DNase I). These two peptides were used as immunogens, both free and conjugated with keyhole limpet hemocyanin (KLH). A rabbit antiserum against the C‐peptide conjugated with KLH (rabbit anti‐C/KLH) and two chicken antisera against the C‐peptide itself (chicken anti‐C) and conjugated with KLH (chicken anti‐C/KLH) reacted well with purified DNase I, blotted onto a transfer membrane after isoelectric focusing in a polyacrylamide gel. A clear isoenzyme pattern identical to that detected with a rabbit antiserum against the parent enzyme (rabbit anti‐DNase I) was observed. Surprisingly, the chicken anti‐C antiserum was found to be a powerful and highly specific tool for the determination of urinary DNase I phenotypes (Kishi et al., Hum. Genet. 1989, 81, 295–297) and was not inferior in any respect to the rabbit anti‐DNase I. These findings show that the selection of immune animal is one of the most important factors for producing an effective anti‐peptide antibody. Unfortunately, the anti‐peptide antisera obtained in this study neither blocked DNase I enzyme activity nor did it bind to the enzyme.
Characterization of gene expression in clinical lung cancer materials by two‐dimensional polyacrylamide gel electrophoresis
Authors: Okuzawa Ken , Franzén Bo , Lindholm Johan , Linder Stig , Hirano Takashi , Bergman Thomas , Ebihara Yoshiro , Kato Harubumi , Auer Gert
Published: May 2005

Fourteen human lung tumors of various histopathological types were subjected to two‐dimensional gel electrophoresis (2‐DE). Samples were prepared for 2‐DE using a nonenzymatic sample preparation (NESP) technique recently established in our laboratory. Variations in the expression of some polypeptides were observed between tumors of different histopathological types. To this end, high expression of β‐tubulin, heat shock proteins 73 and 90, lamin B, and proliferating cell nuclear antigen (PCNA) were observed in small cell lung carcinomas (SCLC). One polypeptide of unknown identity (35 kDa, pI 5.5) was significantly overexpressed in primary lung adenocarcinomas compared with SCLC, squamous cell lung carcinomas, metastatic lung adenocarcinomas from colon and rectum, and normal tissue. The amino acid composition of this polypeptide is presented. In summary, combining the NESP technique and 2‐DE is an effective approach to define tumor‐specific markers.
An examination of the β‐glucosidase (linamarase) banding pattern in flax seedlings using ferguson plots and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis
Authors: Fieldes Mary Ann , Gerhardt Karen E.
Published: May 2005

The reported polyacrylamide gel electrophoresis banding pattern for the main β‐glucosidase (linamarase) component from flax seed consists of five bands, made up of 62.5 and 65 kDa subunits; this component has an estimated molecular weight of 570–670 kDa. The present study used Ferguson plots to estimate the molecular weight of each electrophoretic band, plus two additional bands which were detected. From low to high relative mobility, the seven bands formed a linear series with estimated molecular weights from 1200 to 245 kDa. Each was 160 kDa smaller, and less charged, than the preceding band. This 160 kDa difference between bands did not appear to be consistent with the reported subunit size. Each band produced a corresponding band on sodium dodecyl sulfate (SDS)‐gels. The decreases in molecular weight between the bands on nondenaturing gels and their corresponding bands on SDS‐gels were multiples of the 62–65 kDa value. However, the estimated molecular weights of the SDS bands themselves and of the differences between the SDS bands were, again, not consistent with the proposed subunit size. The results suggest that active forms of this enzyme may contain a second minor component (possibly a 30–35 kDa component) in addition to the 62.5 and 65 kDa subunits.
Protein composition of the human heart: The construction of a myocardial two‐dimensional electrophoresis database
Authors: Jungblut Peter , Otto Albrecht , Zeindl‐Eberhart Evelyn , Pleißner Klaus‐P. , Knecht Mathias , Regitz‐Zagrosek Vera , Fleck Eckart , Wittmann‐Liebold Brigitte
Published: May 2005

Molecular changes occurring in myocardial diseases are not well understood. Proteins, as regulatory molecules, should play an important role in the etiology of these diseases. The method of two‐dimensional electrophoresis (2‐DE) allows the analysis of some thousand proteins with one experiment. An important prerequisite for this kind of investigation is the possibility of identifying the proteins separated by 2‐DE. We resolved 3239 proteins of the human myocardium and tried to identify 33 proteins by amino acid analysis and microsequencing. Twenty proteins were identified by search for the proteinchemical data obtained in the Martinsried Institute Protein Sequence Database. Comparisons of 2‐DE patterns of different size, which were obtained in different laboratories, were performed with the result that proteins identified on a 2‐DE map of one laboratory can be assigned to spots of 2‐DE maps produced by another laboratory. Our results show the usefulness of a myocardial 2‐DE database; they can be used in different laboratories and make it possible to generate a collection of important human myocardial proteins in a 2‐DE database for comparative studies worldwide.
Separation and characterization of rice proteins
Authors: Tsugita Akira , Kawakami Takao , Uchiyama Yotaro , Kamo Masaharu , Miyatake Norifumi , Nozu Yuzo
Published: May 2005

Rice proteins from nine tissues and one organelle (leaf, chloroplast, stem, root, germ, dark germinated seedling, seed, bran, chaff and callus) were isolated and then separated by two‐dimensional gel electrophoresis (2‐DE). The protein spots were characterized according to molecular weight, isoelectric point and partial amino‐terminal sequence. Electrophoresis was carried out by isoelectric focusing (IEF), nonequilibrium pH gradient electrophoresis (NEPHGE) and immobilized pH gradient (IPG) in the first dimension, and by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) in the second dimension. With the aid of nine marker proteins, the patterns of IEF, NEPHGE and IPG 2‐DE gels were graphically combined by computer into a single synthetic image for each tissue, respectively, and these images for the nine tissues and one organelle were again combined into a single 2‐DE image for the integrated rice protein spots. The rice 2‐DE gel image resolved 4892 proteins. About 3% of the spots are characterized by amino‐terminal sequencing.
Use of polyacrylamide gel electrophoresis to detect structural variations in kilobase‐sized DNAs
Authors: Stellwagen Nancy C.
Published: May 2005

The electrophoresis of linear, kilobase‐sized DNA molecules with permuted sequences has been studied in polyacrylamide and agarose gels. Plasmid pBR322, bacteriophage ϕX174, and the SV40 minichromosome were each digested with a series of single‐cut restriction enzymes. The linearized, permuted isomers of all three DNAs exhibit different mobilities in large‐pore polyacrylamide gels, suggesting that all three DNAs contain sites of anisotropic, sequence‐dependent curvature. Various experimental parameters such as acrylamide concentration, crosslinker ratio and buffer composition affect the magnitude of the observed differential mobilities. Band sharpness appears to be optimal in polyacrylamide gels containing 6.9–8.1%T and 0.5–1%C. Only small mobility differences are observed for the linearized, permuted sequence isomers in agarose gels.
The use of coated and uncoated capillaries for the electrophoretic separation of DNA in dilute polymer solutions
Authors: Barron Annelise E. , Sunada Wade M. , Blanch Harvey W.
Published: May 2005

We show that both uncoated and polyacrylamide‐coated capillaries provide separation of large DNA restriction fragments (2.0–23.1 kbp) by capillary electrophoresis in dilute cellulosic polymer solutions. Uncoated capillaries, however, provide significantly better resolution of DNA fragments, particularly when ultra‐dilute polymer solutions are used. This is because electroosmotic flow in uncoated capillaries increases the residence time of DNA in the capillary, without significantly contributing to band‐broadening. At a given field strength and polymer concentration in the buffer, the electrophoretic mobilities of DNA restriction fragments in coated capillaries are virtually identical to those previously measured in uncoated capillaries. It is concluded that the fused silica surface of the capillary does not play a significant role in the mechanism of DNA separation by capillary electrophoresis in uncrosslinked polymer solutions. Thus, the separation of large DNA which has been observed to occur in ultra‐dilute polymer solutions arises primarily from entanglement interactions between the cellulosic polymers and DNA restriction fragments which occur within the bulk of the polymer solution.
Biological activity of prostate‐specific antigen isolated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and electroelution
Authors: Tessmer Uwe , Quack Thomas , Donn Folkert , Leuner Astrid , Dernick Rudolf
Published: May 2005

Human prostate‐specific antigen (PSA), a 33 kDa kallikrein‐like serine protease, occurring in the prostate, in seminal plasma and in blood, was prepared under nonreducing conditions in an enzymatically active form from seminal plasma by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), followed by fast copper staining, electroelution from gel slices and dialysis against isotonic phosphate‐buffered saline (PBS). Enzymatic activity was demonstrated for the first time directly by cleavage of semenogelin, one of the biological substrates of PSA, isolated by the same procedure, i.e. SDS‐PAGE and electroelution, but from seminal vesicle fluid. The purified PSA formed SDS‐stable complexes with the two major extracellular protease inhibitors in blood, α1‐antichymotrypsin (α1‐ACH) and α2‐macroglobulin (α2‐M). PSA isolated under reducing conditions was enzymatically inactive and could not bind to the protease inhibitors α1‐ACH and α2‐M.
Chamber‐specific expression of human myocardial proteins detected by two‐dimensional gel electrophoresis
Authors: Pleißner Klaus‐Peter , Regitz‐Zagrosek Vera , Weise Christoph , Neuß Michael , Krüdewagen Bernhard , Söding Peter , Buchner Klaus , Hucho Ferdinand , Hildebrandt Alfred , Fleck Eckart
Published: May 2005

High resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE), followed by computer‐assisted image analysis (PDQUEST) was used to screen atrial and ventricular protein patterns for quantitative and qualitative differences in protein expression. Myocardial proteins from left ventricular (LV) and right atrial (RA) samples from end‐stage, failing explanted hearts and from a healthy donor heart (control) were separated by 2‐D large gel electrophoresis. Ten RA versus ten LV gels from explanted dilated cardiomyopathic (DCM) hearts were analyzed for quantitative differences in their spot patterns. Of the 197 spots matched to every gel, 40 spots differed significantly in intensity between RA and LV for DCM patients. A larger number of atrial and ventricular gels (20 RA, 20 LV) from DCM patients and from a healthy donor heart (4 RA, 4 LV gels) were analyzed for qualitative differences in protein expression. Three protein spots (SSP 1120: Mr/pI:20.5 kDa/4.6; SSP 1119: Mr/pI:20.6 kDa/4.5; SSP 0117:Mr/pI:20.7/< 4.5) that are present in all RA gels for DCM patients are absent in all LV gels. Two protein spots (SSP 0112: Mr/pI:17.2 kDa,/< 4.4; SSP 0114:Mr/pI:17.6 kDa/< 4.4) occur only in all LV gels but not in the RA gels. These five qualitatively differing spots are identical in DCM patients and in the healthy donor heart. Some of the differing spots were internally sequenced and identified as myosin light chain isoforms (myosin light chain 2, atrial; myosin light chain 2, ventricular; myosin light chain 1, atrial) with the Protein Identification Resource (PIR) accession numbers A44451, S03708, A30881, respectively. Additionally, phosphoglycerate mutase (PIR: JQ0750) and ATP synthase alpha chain (PIR: S17193) were identified. Thus, quantitative and qualitative differences between atrial and ventricular protein patterns were identified by 2‐D PAGE. A characteristic distribution of myosin light chains between atrial and ventricular human myocardium was found using our approach.
Studies on the carbohydrate moieties of the timothy grass pollen allergen Phl p I
Authors: Petersen Arnd , Becker Wolf‐Meinhard , Moll Hermann , Blümke Maren , Schlaak Max
Published: May 2005

Timothy grass pollen was investigated in order to determine the carbohydrate moieties of its major grass group I (Phl p I) and to study its impact on allergenicity. Based on computer calculations one N‐glycosylation site was deduced from the cDNA data of Phl p I. After two‐dimensional polyacrylamide gel electrophoresis, followed by blotting of pollen extract and by use of the monoclonal antibody IG 12 we identified at least six isoallergens of Phl p I with the main spots at a molecular mass of 35–37 kDa and a pI range of 6.5–7.3. Deglycosylation by trifluoromethanesulfonic acid resulted in a decrease of about 2 kDa. Treatment with N‐glycosidase A resulted in a partial deglycosylation, while N‐glycosidase F and O‐glycosidase had no effect. Ten lectins were investigated for their binding to Phl p I components: Aleuria aurantia agglutinin showed strong reactivity (indicating fucose residues), while Galanthus nivalis agglutinin (indicating mannose residues) and concanavalin A (indicating mannose, glucose or N‐acetylglucosamine residues) showed weak binding. By neutral sugar analysis we determined similar contents of the monosaccharides in the isoallergens. In order to study the influence of the carbohydrate structures of Phl p I on IgE reactivity we tested some patient sera for their reactivity with intact and deglycosylated Phl p I. Even though most of the IgE antibodies bind at the protein core, we detected one serum that recognized carbohydrate moieties on the Phl p I.
Multivariate statistical comparison of autoantibody‐repertoires (Western blots) by discriminant analysis
Authors: Zimmermann Christoph W. , Grus Franz‐Hermann , Dux Reinhold
Published: May 2005

A procedure for the quantification and comparison of complex (auto‐) antibody repertoires of many individuals is described. It is based on multivariate statistical analyses of densitometric data of individual staining patterns, in this case Western blots. Implementation of algorithms can be based on standard software and hardware components. This procedure allows (i) the quantitative assessment of the reproducibility and reliability of any electrophoretic procedure, such as for the production of the immunological substrate, (ii) the statistical comparison of any group of staining patterns, e.g., derived from patients with autoimmune diseases or normal controls, (iii) the identification of the bands that contribute most to the differences between such groups, and (iv) the determination whether an unknown individual sample belongs to a known group. A statistical analysis of autoantibody repertoires has important possible applications: it detects spontaneous or therapeutically induced changes of repertoires, it identifies regions of interest, it supports the isolation of relevant antigens, and it will also be helpful in the diagnosis of autoimmune diseases.
Highly sensitive chiral analysis in on‐line combined chiral and achiral media by capillary zone electrophoresis
Authors: Krásenský Simon , Fanali Salvatore , Křivánková Ludmila , Boček Petr
Published: May 2005

A new approach is described for highly sensitive chiral analyses by capillary zone electrophoresis, based on using an on‐line combination of two capillaries filled with either chiral selective or achiral background electrolytes (BGE). Thus, the BGEs are selected in such a way that the first capillary provides optimum chiral selectivity and the second one optimum detection sensitivity. Direct chiral separations of enantiomers of mandelic, m‐methoxymandelic, 3‐phenyllactic and 3‐indolelactic acids served as a model example for testing the approach proposed. The analyses were performed in a BGE containing acetate as a coion and L‐OH‐proline or aspartame as a chiral selector. For high sensitive analyses, an arrangement containing on‐line combined chiral and achiral media were tested in one or two capillaries coupled via a bifurcation block. A detection limit as low as 10−18 moles was reached in the column‐coupling system when the direct chiral separation was performed in the first capillary, filled with 20 mM acetate buffer, pH 4.4, containing 8 mM Cu (II) and 16 mM aspartame (L‐aspartyl‐L‐phenylalanine methylester) and separated enantiomers were detected in the second capillary, filled with 20 mM acetate buffer, pH 3.1. The principle described is of general use in cases where the separation and detection of analytes in question require mutually different BGEs to reach the optimum selectivity and sensitivity, respectively.
High‐resolution sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunochemical identification of the 2X and embryonic myosin heavy chains in complex mixtures of isomyosins
Authors: Rossini Katia , Rizzi Corrado , Sandri Marco , Bruson Adonella , Carraro Ugo
Published: May 2005

In mammals myosin heavy chains (MHC) are polypeptides with a molecular mass of about 200 kDa whose isoforms can be identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and immunochemistry. Electrophoretic analysis is the only method for quantitating MHC profiles in single myofibers and/or cryostat sections of biopsied muscle. We present a method for SDS‐PAGE of adult rat skeletal muscle which resolves MHC into four bands: 1, 2B, 2X, and 2A from the faster to the slower migrating band. Furthermore, embryonic MHC can be also resolved in a complex mixture of isomyosins, e.g. developing or regenerating muscles. The method does not involve preparation of gradient gels or electrophoresis at low temperature. Improved reproducibility is obtained by: (i) modification of the sample buffer; (ii) use of 7% polyacrylamide in the separating gel; (iii) control of pH of running buffer by recirculation or change of the buffer during the run; and (iv) a 24 h run. The procedure is compatible with Coomassie Brilliant Blue, silver and immunoblot staining. Resolution is sufficient to permit transblotting of separated MHC after SDS‐PAGE. The different isoforms are easily identified with monoclonal antibodies. The technique provides an improved method to separate MHC and quantitate MHC2X and MHCemb in complex mixtures of MHC from a few cryostat sections of normal and diseased muscle.
Species identification of cyst and root‐knot nematodes from potato by electrophoresis of individual females
Authors: Karssen Gerrit , Van Hoenselaar Tine , Verkerk‐Bakker Brigitte , Janssen Richard
Published: May 2005

To carry out rapid and reliable species identification of Globodera and Meloidogyne specimens, two electrophoretic methods were modified and adapted for an automated electrophoresis system (PhastSystem, Pharmacia). Proteins of individual Globodera cysts were identified using isoelectric focusing and a sensitive silver stain. Proteins of young single Meloidogyne females were separated using polyacrylamide gradient gels and stained for the isozymes esterase and malate dehydrogenase.
Two‐dimensional gel electrophoresis of nasal and bronchoalveolar lavage fluids after occupational exposure
Authors: Lindahl Mats , Ståhlbom Bengt , Tagesson Christer
Published: May 2005

Human nasal lavage fluids (NLFs), and bronchoalveolar lavage fluids (BALFs) were analyzed with two‐dimensional gel electrophoresis (2‐DE) and the protein patterns were evaluated with a computerized imaging 2‐DE system. With silver staining about 1000 spots were detected in 10 μg samples of NLF or BALF. Both BALF and NLF 2‐DE patterns showed similarities to a reference plasma pattern and about 25 plasma proteins were identified in NLF as well as in BALF. Comparison showed that the levels of albumin and transferrin appeared to be slightly higher in BALF than NLF, while the levels of IgA, IgG and haptoglobin β were higher in NLF than in BALF. In contrast to BALF and blood plasma, NLF contained large amounts of a cluster of acidic proteins (pI 4.5–5.5) with molecular masses of 15–30 kDa. Distinct alterations in the NLF 2‐DE patterns were found in a worker who developed an asthmatic condition with bronchial hyperreactivity after exposure to organic acid anhydrides. After exposure, 14 protein spots were increased and one decreased by a factor of > 3 as compared to the levels before exposure and compared to healthy individuals. This is the first study indicating that 2‐DE of NLF may be used to investigate early changes in airway protein patterns induced by occupational exposure to irritating chemicals.
Characterisation of Haemophilus influenzae proteins by two‐dimensional gel electrophoresis
Authors: Cash Phillip , Argo Evelyn , Bruce Kenneth D.
Published: May 2005

The proteins of nontypable and type b Haemophilus influenzae isolates were characterised using two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). Coomassie Brilliant Blue R‐250 was used for protein detection. Two hundred and twenty eight proteins were resolved from whole cell lysates prepared from a standard nontypable H. influenzae strain (designated HI–64443) when isoelectric focusing was used for the first‐dimensional separation of 2‐D PAGE. When nonequilibrium pH gel electrophoresis (NEPHGE) was used to separate basic proteins in the first dimension, 50 proteins were detected for HI–64443; 20 of the basic proteins detected were considered to be unique for this separation protocol. The apparent molecular weights and isoelectric points were determined for 82 of the proteins resolved for HI–64443. The variation of the proteins from the standard bacterial strain (HI–64443) was determined for nontypable H. influenzae isolates. On the basis of their electrophoretic mobilities, 17.5% of the proteins of HI–64443 were shared by four other non‐typable H. influenzae strains analysed. These data identified both conserved and variable proteins among the nontypable H. influenzae isolates analysed. The results obtained indicated that 2‐D PAGE was able to discriminate nontypable H. influenzae into population clones identified by other procedures. The 2‐D protein profiles obtained for type b H. influenzae strains were similar to those obtained for nontypable H. influenzae strains. The extent of the protein variation observed between type b and nontypable H. influenzae strain was similar to that observed among nontypable strains alone. These data are discussed in relation to the application of 2‐D PAGE as a tool for studies on bacterial epidemiology and for the analysis of the genome structure and gene expression of Haemophilus influenzae.
Single strand conformational polymorphism using capillary electrophoresis with two‐dye laser‐induced fluorescence detection
Authors: Hebenbrock Kirstin , Williams P. Mickey , Karger Barry L.
Published: May 2005

Capillary electrophoresis (CE), using a replaceable polymer matrix and two‐dye laser‐induced fluorescence has been applied to single strand conformational polymorphism (SSCP). Two‐dye laser‐induced fluorescence has been used for improved strand identification over a single‐dye approach. Conditions suitable in the capillary format for rapid separation and high resolution have been explored. The influence of separation parameters such as temperature and matrix composition on separation in SSCP was first determined. Short analysis times allowed for fast screening of optimal separation conditions of the sample. Based on these results, the two strands of a standard 255 bp fragment of the lacI gene were resolved within 25 min with replaceable linear polyacrylamide as a separation matrix. The method was then applied to the detection of different mutations, in the presence of wild type, of a 276 bp fragment of the insulin‐like growth factor 1‐binding protein 1 (IGF1‐BP3) gene.
Probing the inner surface of a capillary with the atomic force microscope
Authors: Barberi Riccardo , Giocondo Michele , Bartolino Roberto , Righetti Pier Giorgio
Published: May 2005

The inner surface of a fused silica capillary for zone electrophoresis was probed by the atomic force microscope (AFM) in the contact mode. Only uncoated surfaces were analyzed, after a simple washing cycle with detergent, NaOH and HCl, and final equilibration in distilled water. Three progressively larger surface areas were probed: 0.5 × 0.5 μ, 2 × 2 μm and 4 × 4 μm. In all cases, it was found that the surface is remarkably smooth, with a median height increasing from 1.3 to 5.6 nm; mean height, from 1.3 to 5.8 nm; root‐mean‐squared roughness, from 0.35 to 1.5 nm; and average roughness, from 0.28 to 0.67 nm (the lower values referring to the smaller area, the larger to the largest area probed). The lowest “peaks” detected are of the order of 1–2 nm; some scattered peaks as high as 16 nm are occasionally found. It is concluded that the inner surface of a capillary is not a serrate or notchy structure, but is indeed quite smooth. Since the average roughness is comprised within the thickness of the diffuse double layer (> 10 nm) existing on the silica wall as a result of silanol ionization (and, in fact, it is on the average considerably smaller), it is concluded that it cannot possibly influence peak shape and contribute to peak decay in an electrophoretic run.
Liposome capillary electrophoresis for analysis of interactions between lipid bilayers and solutes
Authors: Zhang Yanxiao , Zhang Rong , Hjertén Stellan , Lundahl Per
Published: May 2005

Liposomes, which mimic biomembranes, were used as a pseudostationary phase in capillary zone electrophoresis. The decrease in the mobility of an analyte owing to the presence of liposomes reflected interaction between the analyte and the liposomes. Equations were derived to calculate the specific capacity factor Ks (the capacity factor, K′, normalized to the liposome concentration 1 M) from the migration times and to estimate the difference in free energy, Δ(ΔG0), of the weak analyte/liposome interactions. The order of Ks values for the drugs tested was aspirin < salicylic acid < warfarin < sulfasalazine. The peptide TyrGlySerThrProGlyCysCys interacted more strongly with the liposomes (Ks = 10.1 M−1) than did TyrGlySerThrProGlySerSer (Ks = 9.1 M−1). These results were similar to those obtained earlier by immobilized liposome chromatography.
DNA fingerprinting by hybridisation to oligonucleotide arrays
Authors: Southern E. M.
Published: May 2005

Most methods for analysing variation in DNA sequences rely on gel electrophoresis, a method which is difficult to automate and to quantify. Hybridisation to oligonucleotides bound to a solid surface offers alternatives, with the advantage that they can be more readily automated and lend themselves to powerful statistical analytical procedures. In this paper, we discuss a number of different ways in which oligonucleotide arrays could be used for DNA typing for applications in forensic science.
Polymerase chain reaction fingerprinting in fungi using single primers specific to minisatellites and simple repetitive DNA sequences: Strain variation in Cryptococcus neoformans
Authors: Meyer Wieland , Mitchell Thomas G.
Published: May 2005

Minisatellites and simple repetitive DNA sequence motifs are used as conventional oligonucleotide probes in DNA‐hybridization‐based fingerprinting. The same oligonucleotides can be used as single primers in the polymerase chain reaction (PCR) to generate individual PCR fingerprints. In this study, the simple repetitive sequences, (CA)8, (CT)8, (CAC)5, (GTG)5, (GACA)4 and (GATA)4, and a minisatellite core sequence derived from the wild‐type phage M13 (5′ GAGGGTGGCGGTTCT 3′) were used as specific, single primers to amplify hypervariable repetitive DNA sequences during PCR analysis. The potential applications of this technique are demonstrated with clinical isolates of the human pathogenic yeest, Cryptococcus neoformans. PCR fingerprint patterns have remained stable after long‐term in vitro passage (>2½ years to date). Hybridization of the primers to blots of electrophoretically separated chromosomes demonstrated that the target sequences recognized by most of the primers are dispersed through the entire yeast genome. Sequence analysis of the cloned bands obtained by PCR fingerprinting indicated that if the same or extremely similar, inversely oriented tandem repeats are located close to each other, when only one repeat‐specific primer is used in the PCR, the region between these repeats is amplified. PCR fingerprinting has a wide range of current and potential applications to fungi, such as clarifying taxonomic questions, facilitating epidemiological studies and improving the diagnosis of mycotic diseases.
Simple repetitive sequences in the genome: Structure and functional significance
Authors: Brahmachari Samir K. , Meera Gopinath , Sarkar Partha S. , Balagurumoorthy Pichumani , Tripathi Jalaj , Raghavan Sowmya , Shaligram Umesh , Pataskar Shashank
Published: May 2005

The current explosion of DNA sequence information has generated increasing evidence for the claim that noncoding repetitive DNA sequences present within and around different genes could play an important role in genetic control processes, although the precise role and mechanism by which these sequences function are poorly understood. Several of the simple repetitive sequences which occur in a large number of loci throughout the human and other eukaryotic genomes satisfy the sequence criteria for forming non‐B DNA structures in vitro. We have summarized some of the features of three different types of simple repeats that highlight the importance of repetitive DNA in the control of gene expression and chromatin organization. (i) (TG/CA)n repeats are widespread and conserved in many loci. These sequences are associated with nucleosomes of varying linker length and may play a role in chromatin organization. These Z‐potential sequences can help absorb superhelical stress during transcription and aid in recombination. (ii) Human telomeric repeat (TTAGGG)n adopts a novel quadruplex structure and exhibits unusual chromatin organization. This unusual structural motif could explain chromosome pairing and stability. (iii) Intragenic amplification of (CTG)n/(CAG)n trinucleotide repeat, which is now known to be associated with several genetic disorders, could down‐regulate gene expression in vivo. The overall implications of these findings vis‐à‐vis repetitive sequences in the genome are summarized.
Novel separation scheme for capillary electrophoresis of enantiomers
Authors: Guttman András
Published: May 2005

A systematic approach is described for methods development of chiral separations of weak acidic and basic compounds by capillary electrophoresis, using several natural and derivatized neutral cyclodextrins as chiral selectors. Following the methods development scheme suggested here, the appropriate pH of the running buffer as well as the type and concentration of the cyclodextrin is established for the separation of enantiomers. Preselected chiral selectors of β‐cyclodextrin, γ‐cyclodextrin, hydroxypropyl‐β‐cyclodextrin and dimethyl‐β‐cyclodextrin in low and high concentrations, dissolved in low pH, high pH or pH=pK buffers, are employed during the separation method development and optimization. Depending on the type of separation, introduced by Vigh (desionoselective: only the nondissociated; ionoselective: only the dissociated; duoselective: both enantiomers complex selectively), in most instances at least one of the pH/cyclodextrin combinations results in acceptable separation of the solute enantiomers. The viability of the approach is demonstrated through step by step development of chiral separation for several basic and acidic enantiomers.
Interlaboratory reproducibility of yeast protein patterns analyzed by immobilized pH gradient two‐dimensional gel electrophoresis
Authors: Blomberg Anders , Blomberg Lena , Norbeck Joakim , Fey Stephen J. , Larsen Peter Mose , Larsen Martin , Roepstorff Peter , Degand Hervé , Boutry Marc , Posch Anton , Görg Angelika
Published: May 2005

An interlaboratory comparison was conducted on the positional and quantitative reproducibility of yeast proteins resolved by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) using isoelectric focusing with immobilized pH gradient (pH 4–7) in the first dimension. The basic experimental set‐up was as follows: one laboratory prepared and distributed a [35S]methioninelabeled total yeast protein extract (Göteborg, Sweden), another laboratory prepared the IPG strips to be used by all labs in this study (Munich, Germany), the third laboratory (Aarhus, Denmark) circulated the protocols and coordinated the modest attempts to unify them. Samples were run horizontally in the first dimension and vertically in the second. The gels were sent to Göteborg for processing by phosphoimager technology and computerized image analysis (PDQuest), and the 2‐D PAGE resolved proteins were located and quantified automatically. A subset of 470 spots was manually matched in all gels out of an average of 1328 resolved proteins. The positional interlaboratory comparison revealed great pattern reproducibility, the correlation coefficient in no case being less than 0.9994. In absolute terms an average deviation of 2.8 mm (x‐position) and 1.8 mm (y‐position) were obtained for all nine gels (three gels per lab). The interlaboratory comparison of protein quantitation displayed higher variability, and the best correlation coefficient generated was 0.975. An average standard deviation of 34.5% was calculated for protein quantitation including all three labs, a value slightly higher than the intralaboratory variation (range 20–28%) Thus, despite differences in protocols, chemicals and equipment, the immobilized pH gradient technology gave extremely high positional and quantitative reproducibility. This will greatly facilitate the exchange of data and the establishment of multi‐user image‐based 2‐D gel databases.