bluuebunny/crossref_metadata_2025 huggingface dataset:
Biased sinusoidal field gel electrophoresis for the separation of large DNA
Authors: Kotaka Tadao , Adachi Shiro , Shikata Toshiyuki
Published: May 2005

In agarose gel electrophoresis, in a steady, continuous field, it is well known that the mobility μs versus size M relation for linear DNAs (L‐DNAs) can be divided into three regimes: Ogston regime I for small DNAs, where M dependence of μs is weak; entangled but unstretched regime II for intermediate‐size L‐DNAs (of M < 20 kbp), where μs σ M−1 so that efficient fractionation is possible; and entangled and stretched regime III for large L‐DNAs, where M dependence of μs is again weak. Although μs and the regime boundaries can be altered by adjusting the gel concentration Cgel and/or the field strength E, the features of the M dependence of μs are essentially unchanged. As to the effect of DNA topology on μs, we found that in dilute gels (Cgel < 1.0 wt%) coiled, circular DNAs (C‐DNAs) of 2–7 kbp size migrate faster than L‐DNAs of comparable size, while in concentrated gels (Cgel > 1.5 wt%) C‐DNAs migrate much slower than L‐DNAs. To facilitate separation of large DNAs in the regime III range, we proposed biased sinusoidal field gel electrophoresis (BSFGE), which utilizes a sinusoidal field of strength Es and frequency f superposed on a steady bias field of strength Eb. Striking results in BSFGE of low bias (Eb < Es) conditions were that (i) the effective mobility μ at low f (μo) is higher than that of μ∞ at high f, which is equal to the steady field value μs, and (ii) for large DNAs of M > 20 kbp the μ exhibits a minimum μp (pin‐down mobility) at a frequency fp (pin‐down frequency) specific to M, Cgel, and the field strengths in such a way that fp σ M−1 Cgel −1Eb. Esα with α changing from 0 to 2 ∼ 3 at a value of Es dependent on Eb. The μp values appear to fall on the extrapolated portion of the regime II log(μs) versus log M curve. These results are interpreted in terms of the current dynamical models of DNA gel electrophoresis and also with the results of direct observation by fluorescence microscopy on migrating T4dC DNA of 166 kbp in a steady field and under several BSFGE conditions.
Dynamics of DNA under pulsed‐field gel electrophoresis as analyzed from birefringence rise and decay
Authors: Mayer Pascal , Sturm Jean , Heitz Caroline , Weill Gilbert
Published: May 2005

The orientation of DNA induced by electrophoretic transport in agarose gel has been studied by optical birefringence. From its field‐free decay, it is clearly dem‐onstrated that the degree of orientation results from two processes: alignment along the electric field (stretching of the end‐to‐end vector) and elongation of the primitive path in the gel (overstretching). Separation of the two contributions allows the experimental determination of the effective charge per base pair, the gel pore size seen by the reptating molecule, the reptation time, the degree of over‐stretching and the mean relaxation time of the overstretching. Their field and DNA length dependences compare well with theoretical predictions. Similarly, the time at which overstretching presents an overshoot in the rise of the orientation follows closely the predictions of a model based on the evolution of J‐shaped conformations. The recovery of such conformations is studied by a sequence of two pulses with variable delay time. The use of directly measured or extrapolated characteristic times and fields in the design of efficient pulse schemes for pulsed‐field gel electrophoresis is emphasized.
Determination of the isoelectric points of low and high molecular mass ampholytes by capillary electrophoresis
Authors: Klepárník Karel , Šlais Karel , Boček Petr
Published: May 2005

A methodology for a reliable and absolute determination of isoelectric points of amphoteric species by capillary zone electrophoresis is proposed. The principle of this absolute method consists in the application of an additional electroosmotic and/or hydrodynamic flow of a background electrolyte. This allows the measurement of electrophoretic mobilities of substances close to their isoelectric points where the net electric charge is very low and electrophoretic mobility tends to zero. The mobilities are measured at various pH values of the electrolyte so as to find the pH at which the substance moves through the separation column at zero mobility under the action of the additional flow only. The number of required measurements is lowered to the minimum with the help of the iterative procedure based on the Regula Falsi algorithm. The precision of the proposed method is predicted theoretically. The feasibility is demonstrated on real samples. The isoelectric points of histidine, myoglobine and ribonuclease are found from their titration curves.
Selecting DNA fragments for mutation detection by temperature gradient gel electrophoresis: Application to the p53 gene cDNA
Authors: Ke Song‐Hua , Kelly Patrick J. , Wartell Roger M. , Hunter Stephen , Varma Vijay A.
Published: May 2005

Temperature gradient gel electrophoresis (TGGE) and related methods are widely employed to detect mutations in DNA fragments. DNA melting map calculations and GC clamps have been used to enhance the detection of mutations. While generally successful, these methods have not always revealed base changes within a DNA fragment. Previous work suggested that mutations are detected if they are in a DNA's first melting domain, and the melting domain is well separated from final strand dissociation. Two criteria from the DNA melting theory were established to determine when both of these conditions are met. The criteria involve calculating the derivative melting curve as well as the melting map of a DNA sequence. The approach was applied to the cDNA sequence of the human p53 gene. Mutations in the p53 gene are common in human cancers and are generally located in four ‘hot spot’ regions. Calculations indicated that three DNA fragments are needed to detect base substitutions in the four hot‐spot regions. Predicted melting behavior was experimentally tested with eight single base substitutions distributed among the four hot‐spot regions. All mutations tested behaved as predicted and were detected by vertical TGGE. Heteroduplex DNAs formed by melting and reannealing various ratios of wild type and mutant DNA fragments were also examined. Results indicated that point mutations can be detected by ethidium bromide staining from samples containing 10% mutant and 90% wild‐type sequences.
On the kinetics of monomer incorporation into polyacrylamide gels, as investigated by capillary zone electrophoresis
Authors: Righetti Pier Georgio , Caglio Silvia
Published: May 2005

The kinetics of monomer incorporation into a polyacrylamide gel have been studied in a photopolymerization system comprising 100 μM methylene blue in presence of a red‐ox system, 1 mM sodium toluenesulfinate (reducer) and 50 μM diphenyliodonium chloride (oxidizer). A precise assessment of gel point (pc) was obtained in a droplet chamber, in which argon was gently bubbled with a fused silica capillary into the reaction mixture. At pc, 50% (±3) acrylamide was incorporated into the matrix, vs. 80% (±4) N,N′‐methylenebisacrylamide. This incorporation level remained the same when polymerized in the 2–36°C temperature range. Incorporation continued almost linearly for acrylamide up to 80% conversion. The reaction was continued up to 55 min (at 2°C), at which point bisacrylamide had been essentially consumed (>99.5% incorporation) and acrylamide had reacted (95 %). At 2°C, after gelation, the gel became progressively turbid (the Tyndall effect plateauing at 50 min), but it remained fully transparent if, at the gel point, reaction was continued at 50°C. The consumption of the pendant double bonds of Bis followed the progression of turbidity. It is concluded that, by gelation at 2°C, the nascent chains form clusters held together by hydrogen bonds (melting point at 28°C); such clusters are subsequently “Frozen” in the three‐dimensional space as the pendant double bonds in the chains react progressively. Such turbid matrices are more porous and less elastic than when the gel is polymerized at 50°C. This process is similar to the “lateral aggregation” occurring when gels are formed in presence of a polymer in solution (e.g. 10 KDa polyethylene glycol; Righetti et al., Electrophoresis 1992, 13, 587–594).
Pulsed‐field acceleration: The electrophoretic behavior of large spherical particles in agarose gels
Authors: To Kwong‐Yuk , Boyde Tom R. C.
Published: May 2005

In agarose gel electrophoresis, periodically inverting or interrupting the applied field may greatly accelerate the migration of polystyrene microspheres, in a manner varying with pulse times, and the observed zones are made sharper. The particles concerned are just large enough that under constant field they appear not to enter the gel at all or to migrate very slowly: and merely lowering the applied field may also enhance their electrophoretic migration, though to a lesser extent than with field pulsing. These effects may be accounted for by gel mesh flexibility which, varying with the nature of the migrating species, may either help or hinder migration.
Clinical and forensic applications of capillary electrophoresis
Authors: Thormann Wolfgang , Molteni Sarah , Caslavska Jitka , Schmutz André
Published: May 2005

This survey is aimed at giving the readers a short overview of the present state of the art of clinical and forensic applications of capillary electrophoresis. First, the principles associated with electrokinetic capillary separations and instrumentation, sample preparation and solute quantitation are briefly discussed. This is followed by chapters describing the determination of endogenous and exogenous compounds in body fluids and tissue extracts. Finally, a survey of major achievements including reference to fully developed electrokinetic capillary assays is provided. The paper concludes with a brief outlook.
Determination of 2—5A synthetase and 2—5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: Effects of cytokines and comparison with radioenzymatic methods
Authors: Bruchelt Gernot , Büdenbender Manfred , Schmidt Karl‐Heinz , Birk Anette , Treuner Jörn , Niethammer Dietrich
Published: May 2005

2—5A Synthetase and 2—5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2—5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon‐related cytokines (tumor necrosis factor‐alpha and interleukine‐2) on 2—5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2—5A synthetase in these cells. 2—5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2′p5′A2′p5′A (trimeric form of 2—5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus‐32 labeled 2—5A cores was registrated by radioenzymatic assay. Activity of 2—5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2—5A synthetase as well as of 2—5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2′p5′A2′p5′A, respectively.
Separation of enantiomers of N‐acetylcysteine by capillary electrophoresis after derivatization by o‐phthaldialdehyde
Authors: Dette Christoph , Wätzig Hermann
Published: May 2005

The optical purity of N‐acetyl‐L‐cysteine was tested by capillary electrophoresis. The D‐enantiomer can be found down to a ratio of 0.4:99.6 within 4 min without the use of the D‐form or the racemate as a standard. The efficiency was optimized during method development up to a theoretical plate number, N, of several hundred thousand. The migration order and the separation mechanism was explained. The assumed structure of the formed isoindole S‐[2‐(1‐carboxy‐2‐methylpropyl)isoindole‐1‐yl]‐N‐acetylcysteine diastereomers was confirmed and its stability was examined.
Introduction of migration indices for identification: Chiral separation of some β‐blockers by using cyclodextrins in micellar electrokinetic capillary chromatography
Authors: Sirén Heli , Jumppanen Juho H. , Manninen Kirsi , Riekkola Marja‐Liisa
Published: May 2005

Because of the different physiological impact that stereoisomers may have, it is often vital to separate these forms from one another. Because of their structural similarity, the separation is usually difficult to achieve and zones may elute very close to each other. This is a particular problem in capillary electrophoresis, where the repeatabilty of absolute migration times is fairly poor, mainly due to the irreproducibility of the electroosmotic flow. The separation is usually repeatable, however, and when the disturbing effects are eliminated by using a migration index system incorporating two marker compounds the identification of the enantiomers becomes extremely good. Relative standard deviation (RSD) values less than 0.1% for the migration index of each enantiomer were obtained in both intra‐day and day‐to‐day (6 days) studies. The best separation was achieved with the electrolyte solution made of 40 mM borate, 32 mM sodium dodecyl sulfate (SDS), 12 mM β‐cyclodextrin (β‐CD), and 6 mM α‐cyclodextrin (α‐CD) at pH 9.3.
Separations of derivatized amino acid enantiomers by cyclodextrin‐modified capillary electrophoresis: Mechanistic and molecular modeling studies
Authors: Copper Christine L. , Davis Joe B. , Cole Roderic O. , Sepaniak Michael J.
Published: May 2005

The enantiomers of 5‐dimethylamino‐1‐naphthalene sulfonyl (DNS)‐deriva‐tives of selected amino acids were successfully separated using capillary electrophoresis (CE) employing cyclodextrins (CD) as enantio‐selective running buffer additives. A previously described model for retention and chiral recognition in CD‐modified CE is shown to adapt well in this application. Resolution of the isomers is strongly influenced by the type and concentration of cyclodextrin employed, as predicted by the model. Although data indicates differences in the electrophoretic mobilities for some of the completely complexed enantiomer pairs, selectivity generally requires exploiting differences in the amino acid‐CD complexation constants for enantiomer pairs. In this work, the D‐enantiomers exhibit larger formation constants and are complexed to a greater degree (elute first) at moderate CD concentration. When mixtures of amino acids are analyzed, the effects of separation conditions on general elution behavior must be considered or separated enantiomer pairs will co‐elute with other enantiomers. Preliminary results aimed at predicting the strength of DNS‐amino acid enantiomer‐CD interactions based on molecular modeling studies are presented. A statistical mechanical approach to treating computationally derived enantiomer‐CD interaction energies is shown to provide reasonable correlation with separation performance.
Enhanced chiral separation of dansylated amino acids with cyclodextrin‐dextran polymer network by capillary electrophoresis
Authors: Sun Peng , Barker Geoffrey E. , Mariano Greg J. , Hartwick Richard A.
Published: May 2005

A method for the chiral separation of dansyl‐DL‐amino acid mixtures using a dextran (Mr 2 000 000) polymer network containing β‐cyclodextrin (β‐CD) in HPCE was developed. Mixtures of up to seven amino acids could be baseline resolved by this method under neutral pH conditions. Resolution was dependent on the dextran concentration in the polymer network. Temperature effects on the chiral separation were studied. Optimal efficiency and resolution of DL‐enantiomeric pairs of amino acid samples were obtained at 25°C. The resolution of different amino acids used in this work decreased with an increase in temperature.
Pharmaceutical and biomedical applications of capillary electrophoresis/electrochemistry
Authors: Lunte Susan M. , O'Shea Thomas J.
Published: May 2005

The use of capillary electrophoresis/electrochemistry (CEEC) for the analysis of microdialysis samples obtained for pharmacokinetic and neurochemical studies is described, as well as the development of new types of electrodes and waveforms which increase the selectivity of this technique for specific classes of analytes. CEEC with a carbon fiber electrode was employed for the analysis of microdialysis samples. Microdialysis is an in vivo sampling technique that yields very small samples for analysis (less than 1 μL). Therefore, capillary electrophoresis, with its small volume requirements, is an excellent choice for the analytical method. CEEC was used to study the pharmacokinetics of L‐dopa and the release of aspartate and glutamate following a high K+ infusion in the brain. Several modified electrodes which increase the applicability of CEEC in pharmaceutical and biomedical analysis are described. One of these is a gold/mercury electrode which is highly selective for thiols and was used for the determination of glutathione in a rat brain. An alternative method for the detection of thiols employed a chemically modified electrode containing cobalt phthalocyanine. In this case, an electrocatalyst reduces the overpotential of thiols at the carbon electrode and makes it possible to detect them at a much lower and more selective oxidation potential. This electrode was used for the detection of cysteine in urine. The development of pulsed amperometric detection for capillary electrophoresis is also described and is demonstrated by the detection of glucose in blood. Lastly, a method for the detection of peptides based on the formation of a copper complexs and detection at a carbon fiber electrode is discussed.
Multifunctional apparatus for electrokinetic processing of proteins
Authors: Horvath Z. Stephen , Corthals Garry L. , Wrigley Colin W. , Margolis Joel
Published: May 2005

New equipment (the “Gradiflow TM”) has been designed and constructed to provide efficient large‐scale preparative fractionation of macromolecules, based on charge and/or size differences, as well as the concentration of macromolecules and electrodialysis. Examples of its capability are the separation of a mixture of haemoglobin (50 mg) from bovine serum albumin (50 mg) within 15 min (based on charge differences at pH 6.8), the purification of phycoerythrin from a crude extract on the basis of size, and the fractionation of serum proteins into two discrete size classes.
Is gravity on our way? The case of polyacrylamide gel polymerization
Authors: Righetti Pier Giorgio , Bossi Alessandra , Giglio Marzio , Vailati Alberto , Lyubimova Tatyana , Briskman Vladimir A.
Published: May 2005

Potential gravity‐induced deformations of polyacrylamide matrices during gelling were investigated in two different initiator systems based on (i) photopolymerization with 100 μM methylene blue, 1 mM sodium toluene sulfinate (reducer) and 50 μM diphenyliodonium chloride (oxidizer) (photopolymerization) and (ii) chemical polymerization, utilizing the standard persulfate N,N,N,N‐tetramethylethylenediamine. In both systems, it is seen that convective flows are imprinted in the final gel structure above a critical gelling layer thickness, set at ca. 3 mm. In both systems, progressive increments of the solution density, from normodense (density = 1.0) up to isodense with the growing polymer chains (density = 1.3) do not inhibit the appearance of strong convective flows. However, gel inhomogeneities are completely abolished even in 10 mm gelling layers if polymerization is performed in presence of density gradients, notably of sucrose, from 0 to 20%, 0 to 40% and 0 to 60%. Even the shallower gradient (0–20% sucrose) is able to completely abolish convective flows in persulfate‐driven polymerization. It is hypothesized that such disturbances are not created by sedimentation of the growing polymer chains in the gravitational field, but are produced by the reaction exothermality, which produces strong buoyancy‐driven flows. It is additionally demonstrated that persulfate polymerization is sensitive to oxygen absorbed from the top liquid layers, which should be carefully protected by an overlay of organic solvent. Methylene blue‐induced polymerization appears to offer a series of unique advantages over chemical initiation with persulfate.
Analysis of photoreceptor proteins of microorganisms by gradient gel electrophoresis and other biochemical separation methods
Authors: Häder Donat‐P. , Lebert Michael
Published: May 2005

Photoreceptor proteins for photoorientation in microorganisms are usually membrane bound and can be isolated by standard biochemical methods. Three examples are shown: the flagellates Euglena gracilis, Peridinium gatunense and the slime mold Dictyostelium discoideum. The photoreceptor of Euglena is attached to the basis of the flagellum and is composed of at least four chromoproteins which can be separated by gradient sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS‐PAGE) fast protein liquid chromatography (FPLC) and isoelectric focusing (IEF); it contains pterins and a flavin as chromophoric groups. The photoreceptor of Peridinium absorbs in the red wavelength band. Though not yet identified in detail, multiple receptors are probably involved, as indicated by fluorescence spectroscopy. Dictyostelium shows positive and negative phototaxis in its amoebal form and exclusively positive phototaxis in its pseudoplasmodial form. It is still open to discussion whether the two stages use separate photoreceptors. From amoebae two photoreceptor pigments have been isolated, showing an absorption which resembles the action spectrum, one membrane bound with a molecular mass of 45 kDa and one cytoplasmic fraction with a molecular mass of 27 kDa.
Separating field strength, temperature, and pulsing effects in pulsed field electrophoresis
Authors: Mouradian Stéphane , Brumley Robert L. , Smith Lloyd M.
Published: May 2005

The utility of pulsed field electrophoresis for DNA sequencing is investigated. Previous studies have indicated a beneficial retardation of sequencing fragments when pulsed fields are employed. The interpretation of these results is complicated, however, by concomitant variations in electric field strength and/or temperature. Methods are presented here permitting discrimination of such mobility effects due to pulsing, field strength, and temperature. It is demonstrated that under the conditions employed here, observed mobility effects are due to electric field variations rather than pulsing. These conditions thus correspond to the low frequency/small molecule limit. The effect of temperature is estimated from the steady state solution to the heat conduction equation under appropriate boundary conditions. No temperature effect upon mobility is operative in the thin gel system employed, due to the high efficiency of heat transfer. However, it is shown that in conventional gel systems large temperature‐related mobility effects can occur. These methods for dissecting and understanding mobility effects in pulsed field electrophoresis are expected to be of general utility.
A large scale preparative gel electrophoresis separation of α1 and α2 subunits of the voltage‐gated Ca2+ channel from rabbit skeletal muscle
Authors: Nyormoi Okot , Schneider Toni , Smith Robert G.
Published: May 2005

A large‐scale preparative gel electrophoresis method effectively separates individual voltage‐gated calcium channel (VGCC) subunits. with high resolution, starting with up to 4 mg of rabbit skeletal muscle L‐type VGCC complex. Using this method, we separated α1 and α2 subunits of rabbit skeletal muscle VGCC with a high efficiency and with protein recoveries of 83%. The separated α1 and α2 subunits eluted from the gel in a 1:1 molar ratio. The method should be applicable for separating the other VGCC subunits or subunits of other protein complexes.
Protein study of T and B acute lymphoblastic leukemia cell lines
Authors: Mohammad Ramzi M. , Vistisen Kerry , Al‐Katib Ayad
Published: May 2005

Two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to identify cellular proteins in T and B acute lymphoblastic leukemia (ALL) cell lines. Five lines, REH and BALL‐1 of B‐cell lineage, CCRF‐CEM and HPB‐ALL of T‐cell lineage, and a normal Epstein‐Barr virus (EBV)‐transformed line of B‐origin (SKLN1) were studied. The lines were immunophenotyped using flow cytometry and lineage associated monoclonal antibodies. Whole cell lysates of the cell lines were subjected to 2‐D PAGE analyses. 2‐D gels were analyzed with an image scanning computer and the qualitative as well as quantitative differences of the protein patterns were studied. Despite the great similarities in the patterns of the B‐ and T‐gels, three proteins were unique to B‐cell lines, while eight were unique to T‐cell lines. Using cell lines is the first step toward identifying potential markers in ALL and can provide important information regarding the human ALL databases. Whether these proteins are definite markers for B‐ or T‐ALL or are unique to the cell lines studied needs further exploration.
Diode array detection in capillary electrophoresis
Authors: Heiger David N. , Kaltenbach Patrick , Sievert Hans‐Jürgen P.
Published: May 2005

Diode array detection (DAD) in capillary electrophoresis (CE) offers similar advantages over single‐wavelength detection as it does in high performance liquid chromatography (HPLC). Thus, confirmation of compound identity and establishment of peak purity are critical issues in CE, necessitating sensitive and specific detection. With an optimized optical system, DAD yields sensitivity comparable to that of single or variable wavelength detectors. Sensitivity can be further improved three to five times by use of expanded pathlength capillaries employing the so‐called bubble cell. Optimization of optical design, as well as maintenance of spectral fidelity, will be discussed in this work. A variety of applications of CE, and specifically of micellar electrokinetic capillary chromatography (MEKC), with emphasis on quantitative analysis, sensitivity, linearity, spectral identification, and peak purity verification will be presented. The use of spectral information for peak tracking in MEKC method development and for the assessment of purity of electrodistorted peaks will also be illustrated.
Optimized separation of seven corticosteroids by micellar electrokinetic chromatography
Authors: Jumppanen Juho H. , Wiedmer Susanne K. , Sirén Heli , Riekkola Marja‐Liisa , Haario Heikki
Published: May 2005

The corticosteroids studied can be effectively separated by employing micellar electrokinetic capillary chromatography (MECC). The effect of pH, borate concentration and the sodium dodecyl sulfate (SDS) concentration on both the resolution and the selectivity was studied under 15 different experimental conditions. The experimental design was similar to the central composite design approach. Empirical quadratic regression models were derived for analyte migration time, band broadening and analyte velocity. Satisfactory regression fits and coefficients of determination for prediction were obtained with cross‐validated models. The models for analyte migration time and analyte velocity were in good agreement with theory. Modeling of the band broadening seemed to be somewhat more complicated. Optimum conditions for resolution and selectivity were different. This is due to the fact that selectivity studies ignore the electroosmotic and band broadening properties of different electrolyte solutions. However, the study of the selectivity yielded good information about the suitability of the electrolyte systems for the particular separation problem. Although a high solubilizing power of SDS caused the corticosteroids to partition strongly into the SDS micelles, a good separation could be achieved at low SDS concentrations.
Chiral separation of diniconazole, uniconazole and structurally related compounds by cyclodextrin‐modified micellar electrokinetic chromatography
Authors: Furuta Ritsuko , Doi Tadashi
Published: May 2005

Enantiomeric separation of the structural analogs, diniconazole and uniconazole, can be achieved by cyclodextrin (CD)‐modified micellar electrokinetic chromatography (MEKC). The elution behavior of other structurally related compounds was therefore investigated and compared with that in high‐performance liquid chromatography (HPLC) using a CD‐bonded stationary phase. From the results, it was concluded that the benzene ring of the solute plays an important role in formation of CD complexes and chiral recognition in MEKC as well as in HPLC, although the enantioselectivity with the two methods was different. Nuclear magnetic resonance (NMR) data revealed different CD effects on the solute between the MEKC and HPLC models.
Nonaqueous packed capillary electrokinetic chromatographic separations of large polycyclic aromatic hydrocarbons and fullerenes
Authors: Whitaker Kylen W. , Sepaniak Michael J.
Published: May 2005

Polycyclic aromatic hydrocarbons (PAH) and a fullerene mixture (C60/C70) were separated with capillary columns (50 μm ID) packed with a reversedphase packing (octadecylsilica, 3 μm diameter) using electrokinetic pumping. Nonaqueous (acetonitrile modified with methylene chloride or tetrahydrofuran, THF) mobile phases were used for these experiments. The effects of mobile phase composition on such factors as electroosmotic flow, plate height, and capacity factor (K′) are reported. The less polar solvents methylene chloride and THF produced predictable reductions in K′ when used to modify an acetonitrile mobile phase. Large amounts of the less polar modifiers (50% v/v) also resulted in a fourfold decrease in flow rate. This meant that even with a decrease in K′, the retention time increased. Nonaqueous capillary electrokinetic chromatographic (CEC) separations gave efficiencies as high as 160000 plates/m. Use of nonaqueous mobile phases provided small currents which in turn diminished the role of heating effects on efficiency. The nonaqueous system also provided greater solubility for the hydrophobic solutes. A Van Deemter plot for an acetonitrile mobile phase was obtained that exhibited expected trends in plate height with flow rate and K′. Solvent rinses with water and THF are shown to have only small effects on retention and flow rate.
Simulation of reduced band broadening during single‐stranded DNA pulsed field electrophoresis in polyacrylamide gels
Authors: Mayer Pascal , Slater Gary W. , Drouin Guy
Published: May 2005

Using a computer simulation algorithm based on the reptation model, we investigated the effects of pulsed field gel electrophoresis on the separation of single‐stranded DNA molecules in denaturing polyacrylamide gels. Pulsed fields that combine two different field intensities were found to affect the orientation of the reptation tube as well as the electrophoretic velocity, the rate of band broadening, the plate height and the molecular length for which the minimum of mobility was found, in agreement with available experimental results. Due to “memory” effects, pulses alternating between the forward and backward directions can reduce the diffusion constant by many orders of magnitude. Pulses of identical polarity but different intensities reduce the diffusion because stretches of less‐oriented DNA segments are conserved during the migration. We suggest that, for DNA sequencing, pulsed fields of fixed polarity should be used to reduce band broadening and not to overcome band inversion or suppress molecular orientation. We conclude that, using a low intensity, constant electric field will lead to smaller band widths than any pulsed field regime, in a similar length of experimental time, but with less complications.
Protein identifications for a Saccharomyces cerevisiae protein database
Authors: Garrels James I. , Futcher Bruce , Kobayashi Ryuji , Latter Gerald I. , Schwender Brian , Volpe Tom , Warner Jonathan R. , McLaughlin Calvin S.
Published: May 2005

The rapid progress in understanding the genes of the yeast Saccharomyces cerevisiae can be supplemented by two‐dimensional (2‐D) gel studies to understand global patterns of protein synthesis, protein modification, and protein degradation. The first step in building a protein database for yeast is to identify many of the spots on 2‐D gels. We are using protein sequencing, overexpression of genes on high‐copy number plasmids, and amino acid analysis to identify the proteins from 2‐D gels of yeast. The amino acid analysis technique involves labeling yeast samples with different amino acids and using quantitative image analysis to determine the relative amino acid abundances. The observed amino acid abundances are then searched against the current database of 2600 known yeast protein sequences. At present about 90 proteins onour yeast maps have been identified, and the number is rising rapidly. With many known proteins on the map, it will soon be possible to use 2‐D gel analysis to study regulatory pathways in normal and mutant yeast, with knowledge of many the protein products that respond to each genetic or environmental manipulation.
Capillary zone electrophoresis with indirect photometric detection in the visible range
Authors: Malá Zdena , Vespalec Radim , Boček Petr
Published: May 2005

Sensitivity and applicability of commonly used indirect photometric detection in the UV region can be adversely affected by the absorption of the UV light by detected solutes and/or by matrix constituents migrating with them. If the visible light is exploited instead of the UV light, both these disadvantages may be for the most part eliminated, and, moreover, the indirect photometric detection will extend its versatility and sensitivity. Prospects as well as main problems of the approach were tested using inorganic ions as analytes. If organic dyes with a molar light absorption coefficient of the order of 106 mol−1 L m−1 was selected as the light‐absorbing constituents of the background electrolyte, detection sensitivity comparable with the best reported results was reached for anions; for cations, the detection limit was even lower by two orders of magnitude.
Size‐dependent separation of proteins in the presence of sodium dodecyl sulfate and dextran in capillary electrophoresis: Effect of molecular weight of dextran
Authors: Karim Mohammed Rezaul , Janson Jan‐Christer , Takagi Toshio
Published: May 2005

Dextran solutions are widely used as sieving medium in protein analysis by capillary electrophoresis in the presence of sodium dodecyl sulfate. We studied the effect of dextran molecular weight on the separation efficiency using different dextran preparations with wide and very narrow molecular weight distributions, in the range between 1270 and 2 000 000. Migration times and band broadening of proteins were significantly affected by the molecular weight of dextran. Migration times of proteins decreased as molecular weights of the dextrans decreased. Satisfactory separation of all the proteins was possible with all dextrans except those with molecular weights of 70 000 and 23 800 where bovine serum albumin and phosphorylase b failed to be separated. Unexpectedly rapid separation of all the proteins with enhanced resolution could be observed using two dextrans with a narrow molecular weight distribution, with molecular weights of 1270 and 5220. Clearly the use of dextran with higher molecular weight is not the only way to achieve efficient sreparation of proteins. The separation‐mechanism in the presence of the low molecular weight dextrans remains to be made clear in a future study.
Analysis of tyrosinase gene mutations using direct automated infrared fluorescence DNA sequencing of amplified exons
Authors: Oetting William S. , Fryer James P. , Oofuji Yasuhisa , Middendorf Lyle R. , Brumbaugh John A. , Summers C. Gail , King Richard A.
Published: May 2005

The ability to correctly diagnose the molecular cause of genetic diseases is becoming increasingly important in medicine. This requires an efficient method for the analysis of the DNA sequence of specific genes and the detection of mutations in affected individuals. We report a method to determine the mutations responsible for tyrosinase related albinism (OCA1) using a combination of polymerase chain reaction‐single stranded conformational polymorphism (PCR‐SSCP) analysis and direct DNA cycle sequencing using fluorescently labeled oligonucleotides and an automated DNA sequencer based on infrared fluorescence technology. This method allows DNA from several individuals to be sequenced quickly and simultaneously so that the specific location of each mutation and the carrier status of family members can be determined.
Gel polymerization in detergents: Conversion efficiency of methylene blue vs. persulfate catalysis, as investigated by capillary zone electrophoresis
Authors: Caglio Silvia , Chiari Marcella , Righetti Pier Giorgio
Published: May 2005

Four types of detergents are commonly used in biochemical analysis of proteins and polypeptides: neutral, e.g., Triton X‐100, Nonidet P‐40; anionic, typically sodium dodecyl sulfate (SDS); cationic, e.g., cetyltrimethylammonium bromide CTAB), and zwitterionic, e.g., sulfobetaine 3–12 and 3 [(3‐cholamido‐propyl) dimethyl‐ammonio]‐1‐propane sulfonate (CHAPS). These detergents are utilized not only in the protein solubilization step, but also in the polyacrylamide gel matrix in which subsequent electrophoretic separation is carried out. The conversion efficiency of monomers into the growing polymer, in the presence of the four types of detergents, was assessed by capillary zone electrophoresis (CZE) in a micellar system comprising 100 mM SDS, by extracting unreacted monomers from the gel phase and quantifying the peaks separated by CZE. Two different catalyst systems were evaluated: the standard persulfate‐N,N,N′,N′‐tetramethylethylenediamine (TEMED) couple and a mixture comprising methylene blue in presence of the redox couple sodium toluene sulfinate and diphenyliodonium chloride. In the chemically initiated system (persulfate), there was a strong inhibition of polymerization, decreasing in the following order: CTAB > sulfobetaine 3–12 > SDS > CHAPS > Triton X‐100 (e.g., in 10 mM CTAB 100% inhibition was experienced). On the contrary, in photopolymerization (as driven by methylene blue) good conversion of monomers into the growing polymer (> 95 %) was always obtained, independent of the nature of the detergent present in the polymerization step. It is suggested that acrylamide and N,N′‐methylenebisacrylamide (Bis) partition in the detergent micelles, thus being subtracted to equilibrium during polymerization: the order of inhibition in persulfate gels could then be proportional to the monomer partition coefficients. Conversely, the total lack of inhibition in methylene blue gels could be due to the extremely high performance of photocatalysis and/or to its partitioning also inside the detergent micelle.
Field programming to achieve uniform sensitivity for on‐line detection in electrophoresis
Authors: Shear Jason B. , Dadoo Rajeev , Zare Richard N.
Published: May 2005

Under constant applied electric field, an analyte band traverses an on‐line detection zone in an electrophoretic separation at a velocity inversely proportional to the detection time. Because analyte bands migrate with different velocities, the sensitivity of the detection system to different species can vary significantly. One approach for overcoming this problem is to change the applied electric field, E, as a function of the separation time, τ, so that the velocity of an analyte band when it passes through the detection zone is independent of the analyte's electrophoretic mobility. A possible solution is to make E(τ) grow exponentially, but this procedure causes band compression. The preferred solution is to make both E(τ) and the distance between the starting position of the sample and the detection zone increase linearly with separation time. This approach can be implemented with either slab‐gel or capillary electrophoresis.
Polymorphism of pepper (Capsicum annuum L.) seed proteins studied by two‐dimensional electrophoresis with immobilized pH gradients: Methodical and genetic aspects
Authors: Posch Anton , van den Berg Bartel M. , Duranton Claude , Görg Angelika
Published: May 2005

A population of ten morphologically similar inbred lines of pepper (Capsicum annuum L.) has been investigated for polymorphism of seed proteins by two‐dimensional (2‐D) electrophoresis with immobilized pH gradients. To reveal as much variation as possible, both the water‐ and the urea/detergent‐soluble protein fraction were electrophoretically analyzed and subsequently visualized by silver staining. The 2‐D patterns were subjected to computer analysis to be able to establish genetic variation. A high number of the seed proteins were found to be variable as to presence/absence: these were 68 out of 184 reproducible water‐soluble proteins and 34 out of 419 reproducible urea/detergent‐soluble proteins. Comparison of the 2‐D data of the water‐soluble and the urea/detergent‐soluble proteins, which represent the biggest part of all extractable seed proteins, showed that both protein fractions have proteins in common, but the variable proteins found in both fractions were non‐identical. The difference of variability scored in both solubility fractions was discussed. Genetic distances between all pairs of inbred pepper lines were calculated and a genetic tree was constructed. A correlation analysis was carried out to correct for genetic linkage and for secondary modifications, to have a more proper estimate of genetic distances. In both cases the dendrograms showed two distinct genetic groups of five inbred lines. This electrophoretic study was done in order to utilize the genetic distance data in breeding for heterosis. The genetic distance data presented will be used to validate the assumption that there is a higher chance to achieve better hybrid performance when the genetic distance between the parents is as great as possible. From the fact that we found a high level of genetic variability within a population of ten morphologically similar inbred lines, it can be concluded that 2‐D electrophoresis can be efficiently applied in pepper breeding.
A qualitative and quantitative protein database approach identifies individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: An overview of the functional changes associated with the transformed phenotype
Authors: Celis Julio E. , Olsen Eydfinnur
Published: May 2005

A qualitative and quantitative two‐dimensional (2‐D) gel database approach has been used to identify individual and groups of proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes (K14). Five hundred and sixty [35S]methionine‐labeled proteins (462 isoelectric focusing, IEF; 98 nonequilibrium pH gradient electrophoresis, NEPHGE), out of the 3038 recorded in the master keratinocyte database, were excised from dry, silver‐stained gels of normal proliferating primary keratinocytes and K14 cells and the radioactivity was determined by liquid scintillation counting. Two hundred and thirty five proteins were found to be either up‐ (177) or down‐regulated (58) in the transformed cells by 50% or more, and of these, 115 corresponded to known proteins in the keratinocyte database (J. E. Celis et al., Electrophoresis 1993, 14, 1091–1198). The lowest abundancy acidic protein quantitated was present in about 60000 molecules per cell, assuming a value of 108 molecules per cell for total actin. The results identified individual, and groups of functionally related proteins that are differentially regulated in K14 keratinocytes and that play a role in a variety of cellular activities that include general metabolism, the cytoskeleton, DNA replication and cell proliferation, transcription and translation, protein folding, assembly, repair and turnover, membrane traffic, signal transduction, and differentiation. In addition, the results revealed several transformation sensitive proteins of unknown identity in the database as well as known proteins of yet undefined functions. Within the latter group, members of the S100 protein family – whose genes are clustered on human chromosome 1q21 – were among the highest down‐regulated proteins in K14 keratinocytes. Visual inspection of films exposed for different periods of time revealed only one new protein in the transformed K14 keratinocytes and this corresponded to keratin 18, a cytokeratin expressed mainly by simple epithelia. Besides providing with the first global overview of the functional changes associated with the transformed phenotype of human keratinocytes, the data strengthened previous evidence indicating that transformation results in the abnormal expression of normal genes rather than in the expression of new ones.
Two‐dimensional polyacrylamide gel electrophoresis in experimental hepatocarcinogenesis studies
Authors: Wirth Peter J.
Published: May 2005

High resolution two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) in combination with computer‐assisted densitometry was used to analyze sequential changes in polypeptide expression during chemically (aflatoxin Bl; AFB), spontaneously, and oncogene (v‐Ha‐ras, v‐raf, and v‐raf/v‐myc)‐induced experimental rat hepatocarcinogenesis. Two‐dimensional mapping of [35S]methionine and [32P]orthophosphate‐labeled whole cell lysate and nuclear polypeptides revealed subsets of polypeptides specific for each transformation modality in the in vitro rat liver epithelial (RLE) transformation model. Many of the observed changes in whole cell lysate preparations were localized to specific subcellular organelles. Significant alterations in the expression of the extracellular matrix protein, fibronectin, as well as tropomyosin‐ and intermediate filament‐related polypeptides (vimentin, β‐tubulin, cytokeratins 8, 14, and 18, and actin) were observed among the various transformant cell lines. Whereas alterations in the tropomyosin isoforms appeared to be transformation specific, concomitant modulation of intermediate filament expression was related more to the differentiation state of the individual cell lines than to the transformed phenotype. To integrate protein and DNA information of polypeptides believed to be critically involved during cellular transformation, N‐terminal amino acid microsequencing of selected nuclear polypeptides was performed. Preliminary results suggest that N‐terminal blockage of rat liver epithelial nuclear proteins to be minor (∼20%) with sequencing sensitivity of one pmol. These studies extend our on‐going efforts toward the establishment of computerized database of rat liver epithelial cellular proteins (Wirth et al., Electrophoresis, 1991, 12, 931–954) to aid in the delineation of polypeptides critically involved in cellular growth and differentiation as well as transformation.