bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic analysis of serum lipoproteins and apolipoproteins by a combined technique of micro two‐dimensional electrophoresis
Authors: Manabe Takashi , Visvikis Sophia , Steinmetz Josiane , Galteau Marie M. , Okuyama Tsuneo , Siest Gérard
Published: May 2005

Lipoproteins in human serum and in lipoprotein fractions obtained by sequential ultracentrifugation of serum were analyzed by combining three types of micro two‐dimensional electrophoresis (2‐DE) technique to obtain systematic information on lipoproteins and apolipoproteins. The samples were first analyzed under non‐denaturing conditions of electrophoresis to characterize the physicochemical properties of low‐density lipoproteins (LDL) and high‐density lipoproteins (HDL) under physiological conditions. The samples were then analyzed, employing isoelectric focusing without denaturants in the first dimension and sodium dodecyl sulfate (SDS) electrophoresis in the second dimension, to study the process of dissociation of lipoproteins to apolipoproteins. The HDL's dissociated into their constituent apolipoproteins, showing that the charge heterogeneity of HDL is caused by the heterogeneous apolipoprotein content. LDL dissociated into apo B‐100 and minor components. In the third type of micro 2‐DE, employing urea and Nonidet P‐40 in the first dimension and SDS in the second dimension, the lipoproteins were dissociated into their apolipoproteins during the steps of sample treatment, to obtain the information on the contnt of apolipoprotein peptides and their genetic alleles. The combined micro 2‐DE technique will be useful for the systematic analysis of lipoproteins and apolipoproteins in hypo‐ and hyperlipoproteinemic patients.
Influence of flow and diffusion on protein separation in a continuous flow electrophoresis cell: Computation procedure
Authors: Biscans Béatrice , Alinat Philippe , Bertrand Joël , Sanchez Victor
Published: May 2005

Continuous flow zone electrophoresis is an efficient method for the non‐destructive separation of biological materials in a flowing film of buffer solution in which the constituents of a sample are separated according to their electrophoretic mobilities, under the influence of an electric field. This paper deals with modeling of flow structure of the buffer solution taking into account the effect of electroosmosis, Joule heating and thermal free convection and the forced convection of axial flow. Modeling of diffusion is examined in a second part. The modeling equations and the corresponding boundary conditions are solved by finite difference methods. The various parameters affecting the quality of the fractionation are analyzed.
Purification and characterization of three forms of class III alcohol dehydrogenase
Authors: Valkonen Kaija H. , Goldman David
Published: May 2005

We have resolved and characterized three forms of human and rat hepatic class III alcohol dehydrogenase. Separations were carried out in narrow immobilized pH gradients. Both in humans and rats the three forms were visualized by enzyme staining with cinnamol, but not with ethanol. They were insensitive to the inhibitory effect of pyrazole. The isoelectric points were approximately from 6.3–6.4, from 5.9–6.0 and 5.6. Each electroeluted enzyme extract, purified further by analytical isoelectric focusing over the pH range from 5–6 or 6–7, revealed a single band by enzyme and silver staining and by Western blotting followed by avidin‐biotin staining. Polyacryl‐amide gel electrophoresis in the presence of sodium dodecyl sulfate of each extract revealed a single molecular mass species corresponding to class III alcohol dehydrogenase (ADH). All forms of class III alcohol dehydrogenase were recognized by antisera raised against total class III ADH.
Flattening and/or expanding of pH gradients in isoelectric focusing gels exemplified with PhastSystem
Authors: Hackler Rolf , Kleine Tilmann O.
Published: May 2005

A simple method of flattening and/or expanding of pH gradients in isoelectric focusing is described for any pH interval desired: to modify pH gradients near one electrode a paper strip soaked with carrier ampholytes is applied onto the gel close to the opposite electrode. In order to flatten central parts of pH intervals paper strips are applied onto the gel at both electrodes. Conditions and criteria (e.g. amount and pH intervals of carrier ampholytes, width and localization of the paper strip, separation period) for optimization are presented with PhastSystem using ready‐made gels with three different pH intervals and pI marker proteins (Pharmacia). Examples utilizing erythrocyte lysates are presented.
Genetic polymorphism of human α2HS‐glycoprotein: Characterization and application to forensic hemogenetics
Authors: Yuasa Isao , Umetsu Kazuo
Published: May 2005

Human α2HS‐glycoprotein (AHSG) phenotypes were studied by isoelectric focusing of native and desialyzed samples followed by passive immunoblotting. At present a total of 15 different alleles could be distinguished. The usefulness of AHSG polymorphism in the field of forensic hemogenetics was demonstrated by markedly high variability at AHSG locus, reliability of phenotyping results and stability in blood stains.
Moving and stationary boundaries in immobilized pH gradients
Authors: Bjellqvist Bengt , Linderholm Marie , Östergren Karin , Strahler John
Published: May 2005

The relations describing the concentration changes at moving boundaries in a medium containing bound, buffering group are derived for a system which, except for hydrogen and hydroxyl ions, contains one anionic and one cationic mobile constituent. The relations found have been used to calculate concentrations and conductivities in zones developing in immobilized pH gradients. Assumptions used in the calculations as well as conductivity ratios between zones have been experimentally controlled and were found to reasonably agree with expectations. It is also shown how difference in transference numbers between sample droplet and gel will cause concentration and pH changes at the gel‐sample droplet interfaces and it is explained how these changes are related to ionic concentrations in the gel. The high concentration zone generated at one of the interfaces will be transported into the gel. This transport has been numerically simulated and experimentally verified. The low concentration generated at the opposite interface will cause titration impeding sample entrance in the gel through this interface even when the gel contains ions other than H+ or OH− transported towards the interface. The described phenomena explain the dependence of lateral spreading, precipitation at the application site as well as streaking and smearing along sample lanes, on the type and concentration of low molecular weight ions originally present in the gel.
Isoelectric focusing of basic proteins: The problem of oxidation of cysteines
Authors: Altland Klaus , Becher Pia , Rossmann Ute , Bjellqvist Bengt
Published: May 2005

Isoelectric focusing of human globin chains in polyacrylamide gels dried in the ambient atmosphere and rehydrated in the presence of 8 mol/L urea produces artefactual doublets of zones as a result of oxidation by the gel. This oxidation can be avoided in separations of short duration by adding a reducing agent (e. g. 2‐mercaptoethanol or dithiothreitol to the rehydration solution (Altland, K. and Rossmann, U., Electrophoresis 1985, 6, 314–325)). We now demonstrate that the observed zone doublets can be explained by assuming neutralization of the contribution of dissociated sulfhydryl group of cysteine to pI by partial and reversible formation of globin dimers held together by disulfide bridges. Long time separations, requiring e. g. more than 4 h at > 500 V/cm, in pH gradients exceeding pH 7.5, are accompanied by artefactual oxidation from both the atmosphere and the gel matrix. Oxidation from the atmosphere as well as the effect of carbon dioxide can be eliminated by overlayering the gel with paraffin oil. Oxidation from the gel matrix can only partially be inhibited by rehydration of gels in the presence of 2‐mercaptoethanol or dithiothreitol. Nearly complete protection against oxidation by the gel matrix was achieved by adding a permanent supply of 2‐ME to the gel or by adding DTT to the cathodic wick towards the end of the experiment. Alkylation with iodoacetamide or iodoacetic acid resulted in stable globin patterns, which, however, displayed additional artefactual zones. Our experimental data indicate that the polyacrylamide gels function as an electron acceptor for dissociated sulfhydryl groups in proteins, even after pretreatment with strong reducing agents for proteins.
Analysis of human apolipoproteins C by isoelectric focusing in immobilized pH gradients
Authors: Haase Regina , Menke‐Möllers Irene , Oette Kurt
Published: May 2005

Apolipoproteins C are involved in many ways in the metabolism of plasma lipoproteins. Apolipoproteins C from the delipidated VLDL of 35 controls and 165 normo‐and hyperlipoproteinemic patients were analyzed by isoelectric focusing on an immobilized pH gradient, pH 4.0–5.0, with 7 M urea, which raised the apparent pH range to 4.8–5.7. This method is an improvement over conventional isoelectric focusing with carrier ampholytes with regard to both resolution and reproducibility. Due to the high resolution (0.1 pH units per cm) additional apolipoprotein C‐III bands: C‐III0 A1, C‐III0 A2, C‐III1 C and C‐III2 C (the designations A, anodic, and C, cathodic, refer to direction of migration on IEF in relation to the main band) are described for the first time. The possible artifactual nature of these protein bands could be excluded. Cleavage with neuraminidase and peptidases, immunological detection and/or two‐dimensional electrophoresis were used to obtain more information. The additional bands seem, in part, to be hydrolysis products of carboxypeptidase A (C‐III1 C, C‐III2 C). The appearance of C‐III1 C and C‐III2 C was dependent upon the serum triglyceride concentration. The percent distribution of C apolipoproteins in very low density lipoproteins (VLDL) from control serum agreed with previously published data. Apolipoproteins C can also be focused in immobilized pH gradients from VLDL and serum without delipidation.
Identification of mouse brain proteins after two‐dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis
Authors: Eckerskorn Christoph , Jungblut Peter , Mewes Werner , Klose Joachim , Lottspeich Friedrich
Published: May 2005

Two‐dimensional electrophoretic separation and immobilization of proteins onto inert membranes for subsequent amino acid sequence and amino acid composition analysis is described as a rapid procedure for the identification or characterization of proteins from complex mixtures. This method avoids the drawbacks of classical purification and isolation methods which involve time‐consuming operations with low resolution and, often, insufficient yields. Excellent overall yields of minor amounts (in the low μg range) using this method allow for sequence determination of yet inaccessible proteins. Solubilized cell proteins of mouse brain were separated by high resolution two‐dimensionalelectrophoresis and electroblotted onto a siliconized glass fiber membrane. The immobilized proteins were stained with Coomassie Brilliant Blue R‐250, and twelve proteins spots were then submitted to both Edman degradation and amino acid analysis. Proteins were identified by comparison of the experimentally determined amino acid composition with a dataset derived from the Protein Identification Resource (PIR) protein sequence database. Eight out of twelve proteins tested were identified by amino acid analysis and confirmed by N‐terminal sequence determination.
Immunoblotting techniques with picogram sensitivity in cerebrospinal fluid protein detection
Authors: Nespolo Angelo , Bianchi Giorgio , Salmaggi Andrea , Lazzaroni Monica , Cerrato Dario , Tajoli Livia Malesani
Published: May 2005

Agarose isoelectric focusing followed by blotting with nitrocellulose, nylon or polyvinylidene difluoride membranes, and immunochemical detection of cerebrospinal fluid IgG with various combinations of antisera, was evaluated. Polyvinylidene difluoride proved to be an easy‐to‐handle and reliable membrane for protein blotting. Among immunochemical visualization reactions, the most sensitive employed biotinylated goat anti‐human IgG followed by streptavidin colloidal gold conjugate and silver enhancement in 20% w/v urea, allowing a sensitivity of less then 1 picogram IgG/band.
Systematic analysis of the total proteins of a mammalian organism: Principles, problems and implications for sequencing the human genome
Authors: Klose J.
Published: May 2005

High‐resolution two‐dimensional electrophoresis (2‐DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A “complex protein database” is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e. g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods ‐ microsequencing of proteins from spots of 2‐DE protein patterns, and utilization of the relatively short N‐terminal sequences obtained – to produce the corresponding cDNA's of these proteins.
The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence‐specific DNA structure variations
Authors: Diekmann Stephan
Published: May 2005

Curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved seguences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence‐specific DNA twist and wedge angles.
Computer‐assisted evaluation of polydisperse two‐dimensional gel patterns of polysaccharide‐protein conjugate preparations with regard to size and net charge
Authors: Tietz Dietmar , Chrambach Andreas
Published: May 2005

Native Hemophilus influenzae polysaccharide‐protein conjugate particles were analyzed by a two‐dimensional agarose electrophoresis procedure. In view of their preparation by random chemical crosslinking, the conjugates necessarily exhibit a polydisperse two‐dimensional gel pattern which varies depending on the conditions of the particular preparation. The polydisperse patterns were interpreted with regard to the size and surface net charge density of the conjugate on the basis of the extended Ogston model. Data processing was performed by a new program, designated ZWEIDI.DO, written in the language of M‐LAB (modeling laboratory). The program computes particle and gel fiber specific parameters from the positions of standards and unknown(s) on the two‐dimensional gel using a simultaneous linear least‐square curve fitting routine. Based on these calculations, the program serves to compute a nomogram of iso‐size and iso‐free‐mobility profiles. Superimposing these profiles on the gel patterns, the size and free mobility range of the polydisperse conjugate mixtures is obtained. Potentially, the procedure could serve as a tool for quality control in the production of conjugates as vaccines and for the physical characterization of polydisperse subcellular particles and vesicles.
Consecutive radiofluorography and silver staining of two‐dimensional gel electrophoretograms: Application in determining the biosynthesis of serum and tissue proteins
Authors: Kuhn Lotte , Kettman Jack , Lefkovits Ivan
Published: May 2005

The methodology for conventional radiofluorography of two‐dimensional gels, followed by rehydration of the gel and subsequent silver staining, is described. The image obtained by radiofluorography is referred to as biosynthetic image, and the image obtained by silver staining as constitutive image. Since the two images are already in close register (the same gel), reliable identification of polypeptides by the two different assays is possible, and the comparison provides valuable information on the catabolism of each entity. The utility of this procedure is illustrated in experiments involving a labeling with L‐[35S] methionine of an entire mouse. Both serum and tissue samples were analyzed by two‐dimensional gel electrophoresis with the aim of determining several categories of polypeptides in terms of their biosynthetic rates and their composition.
Two‐dimensional electrophoresis aided by personal computer analysis for screening of mutant proteins in inherited diseases
Authors: Yang Rei‐Cheng , Suzuki Yoshiyuki , Tsuji Akihiko
Published: May 2005

A rapid and reproducible method of two‐dimensional electrophoresis was developed for screening of abnormal proteins expressed in fibroblasts from patients with inherited diseases. After silver staining, the electrophoresis gel was subjected to semiautomatic digitizer ‐ personal computer analysis: scanning with an image sensor video camera connected to a digitizer, followed by quantitative determination and statistical analysis with a personal computer. The protein spots analyzed by this method showed quantitative variations of various degrees, particularly in 2 of 247 spots examined. Seven spots were not always detected in control and pathological cells in this study. Slight variations in molecular weight were observed in 3 different spots.
Important parameters in semi‐dry electrophoretic transfer
Authors: Jacobson Gunilla , Kårsnäs Per
Published: May 2005

The efficiency of semi‐dry electrophoretic transfer after sodium dodecyl sulfate (SDS)‐electrophoresis using PhastGel® media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.
Improved detection of lymphocyte membrane proteins in purified form and as a crude mixture using native and denaturing polyacrylamide gel electrophoresis by optimisation of Coomassie Brilliant Blue and silver staining
Authors: Warlow Robert S. , Bernard Claude C. A.
Published: May 2005

Optimised silver staining protocols were devised for the detection of membrane proteins in purified form and as a crude mixture. These were adduced in both sodium dodecyl sulphate (SDS) and native polyacrylamide gel electrophoresis and consisted of ethanol‐acetic acid‐formaldehyde fixation, Coomassie Brilliant Blue prestaining, Rapidfix pretreatment, formaldehyde enhancement and finally ammoniacal silver staining. With these modifications, numerous staining problems of membrane proteins were overcome. These included reduction in background staining, enhanced detection sensitivity in native gels, elimination of negative staining and the avoidance of metallic silver deposition on the gel surface. In overcoming these problems, some factors determining the colour and stainability of membrane proteins in their native state were determined. Both the anionic Coomassie Brilliant Blue dye and SDS detergent improved the sensitivity of silver staining in native gels, and ammoniacal silver was more sensitive than neutral silver, suggesting silver staining to be a charge dependent process.
Use of two‐dimensional gel electrophoresis and autoradiography as a tool in cell biology: The example of the thyroid and the liver
Authors: Lecocq Raymond , Lamy Françoise , Dumont Jacques E.
Published: May 2005

Different applications of two‐dimensional gel electrophoresis and the research strategies that this methodology allows, with examples drawn from our own work on thyroid and liver cells, are described.
Experimental and theoretical description of the isotachophoretic behavior of serum albumin
Authors: Thormann Wolfgang , Mosher Richard A.
Published: May 2005

The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mm, experimental protein plateau concentrations of 1.3–2.3 % w/v are obtained. The computer predictions are approximately half these values.
Numerical analysis of sodium dodecyl sulphatepolyacrylamide gel electrophoretic protein patterns for the classification, identification and typing of medically important bacteria
Authors: Costas Menelaos
Published: May 2005

A numerical analysis of one‐dimensional sodium dodecyl sulphate polyacrylamide gel electrophoretic protein patterns of three separate bacterial groups is described. Similarity between patterns was estimated using a correlation coefficient and is represented diagramatically by the unweighted pair‐group with arithmetic averages method of clustering. Protein patterns were scanned using a laser densitometer interfaced directly to a microcomputer, where calculations were performed semiautomatically with specially written software. A method for the normalization of patterns from different gels is utilized. Whole cell protein patterns clearly distinguished strains representing various groups of Achromobacter. Similarly the two biovars of the Group EF‐4 bacteria could be separated after analysis of partial „background patterns”︁ as could the different species of Providencia. In addition, whole patterns could be used to type the various strains of P. rettgeri. These examples serve to illustrate the power, versatility and potential of the technique when applied to problems of classification, identification and typing of bacteria of medical interest. Its advantages and disadvantages, when compared to other available electrophoretic methodologies, are discussed, as is the future application of such technology to the hospital laboratory.
Blotting efficiency investigated by using two‐dimensional electrophoresis, hydrophobic membranes and proteins from different sources
Authors: Jungblut Peter , Eckerskorn Christoph , Lottspeich Friedrich , Klose Joachim
Published: May 2005

Purification and chemical characterization of protein may be achieved by combining two‐dimensional electrophoresis (2‐DE) and microsequencing or amino acid analysis. To enable this combination, the protein has to be transferred as completely as possible from the gel into the sequencer. In this study hydrophobic membranes were used as support for the transfer and proteins were transferred from the gels onto the membranes by semidry blotting. Blotting conditions were optimized to obtain high blotting efficiencies for as many proteins of a complex 2‐DE pattern as possible. Under optimized conditions, blotting efficiencies between 60 % and 100 % were obtained for five marker proteins; the mean values from four regions of a 2‐DE pattern from 29 unknown proteins of a complex protein mixture from mouse brain were between 60 % and 79 %. The four commercially available hydrophobic membranes that were compared showed only slight differences in protein amount on the membranes after blotting for whole protein patters, whereas single proteins occurred with higher amounts on either one or the other membrane. The results of the blotting optimization allowed us to suggest a blotting mechanism with which systematic improvement of the blotting conditions is possible for problematic proteins.
An improved scheme for the identification of antigens recognized by specific antibodies in two‐dimensional gel electrophoresis and immunoblotting
Authors: Shen Horng‐Der , Choo Kong‐Bung , Lin Win‐Ling , Lin Run‐Yee , Han Shou‐Hwa
Published: May 2005

This paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of two‐dimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred to a polyvinylidene difluoride membrane which does not shrink or change dimensions in organic solvents. The efficiency of transfer and the localization of sample proteins on the membrane are checked and recorded by staining the blotting membrane with Fast Green FCF and recording the profile on a transparency. After blocking and the immunoassay, the results are recorded by photography. The sites of immune reaction are marked and the same membrane is restained briefly with Coomassie Brilliant Blue R‐250 for the protein profile. Thus antigens in complex mixtures, recognized by antibodies of interest, can easily be identified from the restained membrane. If the whole protein profile is not well demonstrated, when used in combination with the profile recorded on the transparency, spots appearing on the restained membrane can still be used as useful landmarks in the final unequivocal antigenic identification. This improved scheme circumvents problems arising from membrane shrinkage and difficulties in accurately matching immunoreactive spots by conventional procedures and thus provides an accurate, simple and fast approach in the identification of antigens after immunoblotting.
Development of a database of amino acid sequences for human colon carcinoma proteins separated by two‐dimensional polyacrylamide gel electrophoresis
Authors: Ward Larry D. , Hong Ji , Whitehead Robert H. , Simpson Richard J.
Published: May 2005

The tandem use of preparative two‐dimensional polyacrylamide gel electrophoresis (2‐DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R‐250, and selected protein spots were excised and subjected to Edman degradation. Our résults demonstrate that overall sequence yields in the 3–20 pmol range can be achieved on protein spots from four identical 2‐DE gels; approximately 150–200 μg of total protein was applied to a single 2‐DE gel. An approximate two‐fold increase in sensitivity of phenylthiohydantoin‐amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin‐amino acid derivative. N‐Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of protein of known structure listed in the available protein sequence databases. Approximately 40 % of the electroblotted proteins did not yield N‐terminal sequence information, presumably because they had blocked N‐termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2‐DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one‐dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.
Influence of carbohydrate moieties of human serum transferrin on the determination of its molecular mass by polyacrylamide gradient gel electrophoresis and staining with periodic acid‐Schiff reagent
Authors: Riebe Dieter , Thorn Werner
Published: May 2005

The influence of carbohydrate moieties of transferrin (Tf) on the determination of its molecular mass (MM) by polyacrylamide gradient gel electrophoresis (PAGGE) was investigated. Iron‐free native human serum transferrin (Tf) of 99% purity and partly or completely carbohydrate‐ and N‐acetylneuraminic acid (NANA)‐free molecule forms were analyzed. The MM differences before and after enzymatic cleavage were found not to agree with the theoretical difference. From amino acid and carbohydrate analysis the MM of Tf was determined to be 79 570 Da whereas by denaturing and nondenaturing PAGGE MM of 77 000 Da ± 1000 Da were found. After enzymatic cleavage of the two carbohydrate chains of Tf the difference between the calculated MM and the value reported in literature increased to 7000 Da (nondenaturing PAGGE) and 9200 Da (denaturing PAGGE). Following enzymatic cleavage of the 4 NANA molecules (MM 1237, Da) we obtained the relatively largest difference between the value given in the literature and that determined by PAGGE, namely MM 3300 Da on nondenaturing and 4000 Da on denaturing PAGGE. The differences due to the removal of the other carbohydrates were negligible. In addition we tested the periodic acid‐Schiff reagent to stain iron free Tf, containing different carbohydrate residues. The shortest carbohydrate moiety necessary for Tf staining corresponds to two identical carbohydrate chains of the structure (Asn)‐GlcNAcGlcNAc‐α‐Man‐(α‐Man‐)‐α‐Man.
Detection of conformational and net charge differences in DNA‐protein complexes by quantitative electrophoresis on polyacrylamitie‐agarose copolymer gels
Authors: Orbán Lászó , Chrambach Andreas , Zwieb Christian , Adhya Sankar L.
Published: May 2005

The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 by pallindromic DNA sequences, differing in four of the base pairs (Fig. 1). OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by “quantitative gel electrophoresis”. By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA‐GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR‐O1 and GalR‐OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA‐protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp). Thus, the apparently larger size of the GalR‐O1 complex at the center of DNA, compared to that of the GalR‐OE complex, supports the previous conclusion that the Gal repressor induces a larger degree of bending in O1 DNA than in OE DNA. DNA fragments bearing OE and O1 when complexed with the Gal repressor show convex Ferguson plots at polyacrylamide concentrations below that of gelation and in the presence of 0.5 % agarose, while the plots of the corresponding free 155 bp DNA fragments are linear in that range. The curvature presumably reflects the size increase concomitant with complex formation, since DNA larger than 155 bp also exhibits that curvature on polyacrylamide stabilized by nonrestrictive agarose gels.
Alcian blue fixation allows silver staining of the isolated polysaccharide component of bacterial lipopolysaccharides in polyacrylamide gels
Authors: Corzo Javier , Pérez‐Galdona Ricardo , León‐Barrios Milagros , Gutiérrez‐Navarro Angel M.
Published: May 2005

The effect of the cationic dye Alcian Blue on the silver staining of bacterial lipopolysaccharide and its polysaccharide and lipid A portions in polyacrylamide gels was investigated. The polysaccharide was only stained when the gel was previously treated with the dye. The polysaccharide moiety was found to be responsible for the lipopolysaccharide staining with silver, whereas the lipid A seemed unimportant. Treatment with Alcian Blue may prove useful to detect hydrophilic components of lipopolysaccharide samples that could no be stained by the usual silver staining procedures.
An improved method combining two electrophoretic procedures: Application to the separation of lens α‐crystallin isoforms
Authors: Arruti Cristina , Chifflet Silvia
Published: May 2005

Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS‐PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat‐bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of α‐crystallins of young and embryonic quail lenses.
Electrophoresis with intermittent scanning of the migration path: Detection of resolution within shortened time
Authors: Deml Mirko , Garner Mark M. , Chrambach Andreas
Published: May 2005

Intermittent optical scanning (by detection of optical density or fluorescence) of the electrophoretic migration path was applied to the resolution of two dyes under an arbitrary set of conditions. Scanning at 5‐min intervals allows for detection of resolution between the two zones at least 3 times faster than conventional automatic zone detection employing a detector at the end of the migration path. This result promises that replacing stationary by mobile detectors in general would result in a substantial time saving for automated detection of electrophoretic zones.
Why can we not sequence thousands of DNA bases on a polyacrylamide gel?
Authors: Slater Gary W. , Drouin Guy
Published: May 2005

Pulsed fields have been remarkably useful at extending the range of DNA molecular sizes that can be separated on agarose gels by controlling the field‐induced molecular orientation that often limits the resolution of large molecules. Unfortunately, the same approach seems to be much less effective for DNA sequencing on polyacrylamide gels. We present an experimental and theoretical (modelling) study of DNA sequencing which shows that molecular orientation is indeed not the main limiting factor for sequencing devices that use moderate field intensities and polyacrylamide as a separating matrix. We examine the interplay between electric field intensity, molecular size and resolution, and we suggest different approaches to increase the resolution limit of standard and automated sequencing gels. The theoretical limits of high‐field electrophoretic sequencing are also discussed. We conclude that new ideas will be needed to go beyond one kilobase.
Localization of separated protein bands in unstained electrophoresed polyacrylamide gradient slab gel
Authors: Arora D. Jit S.
Published: May 2005

Polyacrylamide gel electrophoresis has become the most widely used separation method in biological science. Once electrophoresis is complete the protein bands must be localized prior to excision. A zig‐zag gel cutter is described which cuts a strip of gel from the side of a slab gradient gel or a gel of uniform concentration in peaks and valleys. The location of the protein of interest is determined by counting the number of peaks on the stained side strip. The portion of the unstained gel corresponding to the same count (number of valleys) is cut to recover the protein of interest from the main gel for further manipulations.
Apolipoprotein E phenotyping by isoelectric focusing in immobilized pH gradients and silver staining
Authors: Cartier Régine , Sassolas Agnès
Published: May 2005

A simple and high resolution procedure of apoprotein E (apo E) phenotyping by isoelectric focusing with immobilized pH gradients and silver staining is described. This method needs delipidated very low density lipoproteins (isolated from 1 mL of serum) but obviates immunoblotting as well as neuraminidase treatment in routine applications because the sialylated forms are clearly separated. Immunoblotting (with polyclonal and monoclonal anti‐apo E antiserum), cysteamine and neuraminidase treatment, and pI markers allowed the localization of three main alleles, ξ2, ξ3, ξ4 and the detection of variants or rare alleles (6/450 determinations). The serum amyloid A (SAA) apolipoproteins (SAA1, SAA2) could be characterized unequivocally (especially with E3 and E4). Silver staining proved more sensitive than Coomassie Brilliant Blue and needs only 5 μg of protein in the sample. The results of 403 normo‐or hyperlipidemic patients are shown. In the group of 191 normolipidemic patients (cholesterol < 6.40 mmol/L triglycerides < 2 mmol/L), the relative frequency of the ξ3 allele (0.83) is higher than in other reports on Caucasians (about 0.77) whereas the ξ4 allele is lower. As previously described, we find a high frequency of the 4/3 phenotype in hypercholesterolemia and 3/2 in hypertriglyceridemia. The high frequency of the E2/E2 phenotype, usually associated with hyperlipidemia, and variants in complex hypertriglyceridemia makes the apo E phenotyping necessary in many cases of dyslipidemias.
Calculation of restriction fragment lengths by image processing
Authors: Holmlund Gunilla , Karlberg Knut , Gustavsson Bengt , Lindblom Bertil
Published: May 2005

The image processing system, LabEye Profile, calculates the size of DNA restriction fragments according to the formula MD = f(x); x = 1/log bp. The migration distances (MD) of known fragment sizes are interpolated by the use of cubic splines. Cubic splines are also used to correct for gel distortions. A comparison to the Biotrac system, which uses the reciprocal method, gives almost identical standard deviations for calculated sizes compared to known sizes, 0.88% for LabEye Profile and 0.89% for Biotrac. The measurement error for LabEye Profile is 0.24% of the size of the fragments.
Elimination of sample diffusion and lateral band spreading in isoelectric focusing employing ready‐made immobilized pH gradient gels
Authors: Loessner Martin J. , Scherer Siegfried
Published: May 2005

A simple procedure for the elimination of lateral sample as well as band spreading in precast, ready‐made immobilized pH gradient gels is described. Round or rectangular holes are punched in the dry polyacrylamide gels prior to rehydration. The generated wells proved suitable for application of samples containing surfactants such as Nonidet P‐40 or 3‐[(3‐cholamidopropyl) dimethylammonio]‐1‐propane‐sulfonate (CHAPS). Lateral band spreading and precipitation of samples containing up to 9.5 M urea could be completely eliminated by this method.
Studies of DNA bending and flexibility using gel electrophoresis
Authors: Harrington Rodney E.
Published: May 2005

Gel electrophoretic methods have become established as primary tools in the study and elucidation of sequence‐directed curvature both in free DNA and in the operator DNA of several site‐specific nucleoprotein complexes. Results using them have been generally consistent with physical methods sensitive to DNA structure and conformation in those instances where direct comparisons can be made, and in a number of cases, gel methods have provided unique information not presently available from other techniques. Two basic strategies have been used: one based upon anomalous gel mobility effects; and a second based upon cyclization properties of curved DNA. Within each of these categories, various approaches have been used, some of which can lead, in favorable cases, to quantitative estimation of bending angles. In this review, the various gel‐based methods that have been used to date are critically discussed and the qualitative and quantitative information that can be obtained from them is evaluated. A number of possible structural models for DNA curvature are described and a distinction is drawn between static or fixed bending and bending due to anisotropic flexibility at specific sequence loci. The importance and roles of gel electrophoretic methods in providing experimental approaches to this question are discussed. It is suggested that both static curvature and anisotropic flexibility in operator DNA may provide much of the basis for indirect readout of sequence information by specific sitebinding regulatory proteins.
Capillary zone electrophoresis of p‐aminobenzoic acid derivatives of aldoses, ketoses and uronic acids
Authors: Grill Eduard , Huber Christian , Oefner Peter , Vorndran Andreas , Bonn Günther
Published: May 2005

Aldoses, ketoses and uronic acids were derivatized with p‐aminobenzoic acid and separated as their borate complexes by capillary zone electrophoresis, using a capillary tube of fused silica containing 150 mM borate buffer, pH 10.0, as carrier. The electrophoretic mobilities of 22 carbohydrates were determined and found to increase with increasing stability of the borate complexes formed. Besides the number of hydroxyl groups and the presence of substituents, complex stability depended most strongly on the configuration of the three vicinal hydroxyl groups at C2, C3 and C4. On‐column UV monitoring at 285 nm allowed the detection of glucose with a lower mass detection limit of 15 fmol and a concentration sensitivity of 4 μM. Reproducible quantification of carbohydrates was achieved at least in the concentration range of 0.1–10 mM in reaction solutions by the relative peak area method, using cinnamic acid as internal standard. The method was applied successfully to the determination of the monosaccharide composition of polysaccharides extracted from Radix althaeae.
Numerical simulation of protein separation by continuous‐flow electrophoresis
Authors: Clifton Michael J.
Published: May 2005

Continuous‐flow electrophoresis is a process for separating protein mixtures on a preparative scale. Its resolution is determined by the migration distance at the collection plane and by the fineness of the filament occupied by each protein species. Filaments undergo spreading due to a number of different phenomena, among which electrokinetics and electrohydrodynamics are known to be important. In the first of these, differences in migration velocity between the ionic species give rise to local variations in pH and electrical conductivity near the protein filament. In the second, the local change in electrical conductivity distorts the electric field, thus inducing shear stress in the liquid and creating a local flow pattern. A numerical model has been developed to describe these phenomena when two proteins are being separated.
Two‐dimensional separations of peptides and proteins by comprehensive liquid chromatography‐capillary electrophoresis
Authors: Larmann John P. , Lemmo Anthony V. , Moore Alvin W. , Jorgenson James W.
Published: May 2005

Analysis of biological samples is problematic because of their complex composition. Reversed phase liquid chromatography (RPLC), size exclusion chromatography (SEC), and, more recently, capillary zone electrophoresis (CZE) are routinely used for the analysis of these samples, but are eventually limited because they are one‐dimensional (1‐D) methods. As sample complexity increases, the separation efficiency necessary to resolve a large number of sample components in one dimension becomes prohibitively high. A solution to this problem has been to use a two‐dimensional (2‐D) approach. Each dimension in a 2‐D separation relies on a different separating mechanism. By expanding the separation into two dimensions, sample components unresolved in the first dimension can often be separated in the second. This circumvents the requirement for extremely high efficiencies in either dimension. Two‐dimensional slab gel electrophoresis has been used successfully in this area, but a more instrumental approach is desired. In this paper we describe three coupled‐column approaches to 2‐D separations. First, microcolumn SEC‐CZE is explored as a means of 2‐D protein analysis. Next, RPLC‐CZE is investigated for analysis of peptides in tryptic maps. Finally, RPLC is coupled with fast CZE (FCZE), a unique form of CZE analysis, for fast 2‐D analysis of peptides. Details of the instrumentation used in these 2‐D systems will be presented along with the results of some typical 2‐D analyses.
Application of high‐performance capillary electrophoresis to the analysis of H1 histones
Authors: Lindner Herbert , Wurm Martin , Dirschlmayer Arnold , Sarg Bettina , Helliger Wilfried
Published: May 2005

High‐performance capillary electrophoresis for the separation of rat testis H1 histone variants and their phosphorylated modifications is described. The influence of buffer pH, hydroxypropylmethyl cellulose, and buffer concentration has been investigated. Under optimized conditions (500 mM phosphate buffer, pH 2, 0.03% hydroxypropylmethyl cellulose) using an uncoated capillary, eight H1 histone subfractions, including two H1° histones and H1t and their phosphorylated modifications, are resolved. Application of capillary electrophoresis to the separation of H1 histones provides an important new alternative to high‐performance liquid chromatography (HPLC) and traditional gel electrophoresis.
Potential and limitations of an optically active crown ether for chiral separation in capillary zone electrophoresis
Authors: Kuhn Reinhard , Wagner Joseph , Walbroehl Yvonne , Bereuter Thomas
Published: May 2005

Capillary zone electrophoresis using optically active 18‐crown‐6 tetracarboxylic acid (18C6H4) as chiral selector was studied for the enantiomeric separation of primary amines. From the separation of a variety of pharmaceutical drug substances, amino alcohols and amino acids, conclusions could be made concerning the influence of the chemical structure of the analytes on the separation. In addition, the effects of experimental parameters such as pH, proportion of organic modifier and buffer composition on the separation are discussed. A synergistic effect obtained by the joint application of 18C6H4 and a cyclodextrin was exploited to resolve analytes which were separated neither by the crown ether nor by the cyclodextrin.