bluuebunny/crossref_metadata_2025 huggingface dataset:
Viscous effects in capillary electrophoresis: Theory and experiment
Authors: Schure Mark R. , Murphy Robert E.
Published: May 2005

Simple theory which relates the viscous effects of additives used in capillary electrophoresis (CE) to resolution and resolution per unit time are developed for small molecule separations in the absence of sieving effects. The resolution theory shows that there is no advantage to using viscous additives for resolution improvement unless either a binding energy difference exists between the solutes and additive, or viscous effects cause a reduction in the vector sum of electrophoretic and electroosmotic velocities when these are of opposite sign. In general, increasing the viscosity is shown to result in a loss of resolution per unit time. Theory demonstrates that in cases where only binding is considered to be present, the maximum resolution increase is found to be at relatively small amounts of additive. In addition, specific regions of the electrophoreogram may demonstrate resolution enhancement at a specific concentration while other regions exhibit a resolution decrease, as compared to no additive present. CE separations of the components in a Triton surfactant, conducted using a polyethylene glycol (PEG) additive, demonstrate that improved resolution for some of the peaks exists at a specific PEG concentration. These results are mostly explained through the reduction in electroosmotic flow velocity that takes place through additive adsorption to the capillary wall, although some binding is present.
Band width measurement in automated gel electrophoresis apparatus: DNA dispersion in a discontinuous system and in a single buffer
Authors: Yarmola Elena , Chrambach Andreas
Published: May 2005

Recent commercial introduction of automated gel electrophoresis apparatus allows for band width measurements during electrophoresis and therefore promises to open up the exploitation of band width and shape for the physical characterization of charged macromolecules in the same manner in which to date quantitative gel electrophoresis had exploited electrophoretic mobility at multiple gel concentrations. The measurements demonstrate decreased band width and therefore increased resolving power for a discontinuous buffer system compared to Tris‐borate EDTA buffer. The dispersion coefficients (D′ = (σ2 ‐σ20)/t) of homogeneous DNA components appear to decrease with gel concentration when either the field strength or the DNA length is small, and increase with gel concentration when these are large. This contrasting response of D′ to increasing gel concentration is presumably due to DNA stretching, which increases in proportion to DNA length and field strength, and to the progressive orientation of agarose with increasing field strength.
Determination of amino acids by on‐line capillary electrophoresis‐electrospray ionization mass spectrometry
Authors: Lu Wenzhe , Yang George , Cole Richard B.
Published: May 2005

On‐line capillary electrophoresis‐electrospray ionization mass spectrometry (CE‐ESMS) has been used for the separation and detection of amino acid mixtures. Four natural amino acids, histidine, tryptophan, phenylalanine, aspartic acid, and a tripeptide, glutathione, were separated and determined. Protonated molecules were detected in the CE‐ESMS mode with detection limits of about one pmol. Optimum CE‐ESMS operating conditions for amino acid analysis were determined employing acetic acid solutions as CE electrolytes. The examined parameters included capillary diameter (50–100 μm internal diameter), applied separation voltage (20–30 kV), and concentration of electrolyte (10–60% acetic acid). Stable working conditions were maintained when the CE currents were less than 18 μA. The use of electrolyte solutions such as those described here instead of true buffer solutions may have advantages for CE‐ESMS systems which employ a “sheathless” interface.
Capillary electroseparations of enkephalin‐related peptides and protein kinase A peptide substrates
Authors: Thorsteinsdóttir Margrét , Beijersten Ingegerd , Westerlund Douglas
Published: May 2005

The separations of enkephalin‐related peptides and protein kinase A peptide substrates, with the common structural feature ‐Arg‐Arg‐X‐Ser‐Val‐, were studied in micellar electrokinetic chromatography (MEKC) systems and compared with the capillary zone electrophoresis (CZE) mode. The influence of the magnitude and the direction of the electroosmotic flow on the selectivity was studied. Reversed electroosmosis was obtained by adding a hydrophobic amine, dimethyldodecylamine, to the background electrolyte; the amine forms cationic micelles with a low critical micelle concentration (0.3 mM). The neutral micellar agent, Brij 35, competes with the amine for adsorption sites on the capillary surface decreasing the reversed electroosmosis. In such a system, mixed cationic micelles are formed to which the peptides were not distributed at low pH, but an improved resolution was obtained due to the effects on electroosmosis. In systems containing the less hydrophobic amine dimethyloctylamine, in which probably no mixed micelles are formed, an improved separation of protein kinase A peptide substrates was obtained due to distribution to Brij 35 micelles. In separations of enkephalins, a high pH gave very low efficiencies due to surface‐analyte interactions, and the best CZE separations were obtained at low pH. Changes in migration order were observed in the pH range 2–3, possibly due to differences in peptide pKa values or conformation changes of the peptides. The enkephalins were only to a small extent distributed to the Brij 35 micelles, but this improved the separation at pH 2 compared to the CZE mode.
One‐ and two‐dimensional histone separations in acidic gels: Usefulness of methylene blue‐driven photopolymerization
Authors: Rabilloud Thierry , Girardot Valérie , Lawrence Jean‐Jacques
Published: May 2005

We tested the recently introduced methylene blue‐toluene sulfinate‐diphenyl‐iodonium polymerization system in order to prepare acetic acid‐urea‐Triton X‐100 gels for histone separations. When compared to standard persulfate‐based initiators, this system exhibited several advantages. First, the polymerization proceeds at a much faster rate but is easily controlled since it is light‐dependent. Second, no prerunning of the gel was required, since there is no oxidizing molecule able to introduce artifacts. Moreover, this procedure produces gels presenting cleaner backgrounds with silver staining. The ability of the procedure to carry out high resolution two‐dimensional electrophoresis of histones was also examined. High resolution two‐dimensional gels, using photopolymerized acidic gels as the first or second dimension were obtained.
Assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis
Authors: Michaud Dominique , Cantin Line , Raworth David A. , Vrain Thierry C.
Published: May 2005

A method for assessing the stability of cystatin/cysteine proteinase complexes using mildly‐denaturing gelatin‐polyacrylamide gel electrophoresis (gelatin‐PAGE) is described. As suggested by the use of well‐known cystatins (human stefins A and B, and oryzacysatins I and II) and the plant cysteine proteinase papain, the ability of cystatin/cysteine proteinase complexes to remain stable during electrophoresis is associated with the degree of affinity between the enzyme and the inhibitor (and inversely associated with the Ki values), at least with the disulfide bond‐lacking cystatins. Complexes with Ki values ≥ 10−8 M (weak interactions) are partly or completely dissociated under the conditions used, while those with lower Ki values (strong interactions) remain stable. As shown by the differential effects of two plant cystatins, oryzacystatins I and II, against a cysteine proteinase present in crude (complex) extracts from a plant pest — the two‐spotted spider mite (Tetranychus urticae Koch), the gelatin‐PAGE procedure is suitable for studying the ability of cystatins to form highly stable complexes with cysteine proteinases, without the need for prior purification steps. Considering the well‐recognized potential of proteinase inhibitors for pest and pathogen control, this analytical approach will be useful for rapidly assesing the respective potential of various cystatins for protection of plants, animals, and humans.
Analysis of ε‐N‐2‐furoylmethyl‐L‐lysine (furosine) in dried milk by capillary electrophoresis with controlled electroosmotic flow using N,N,N′,N′‐tetramethyl‐1,3‐butanediamine in the running electrolyte solution
Authors: Corradini Danilo , Cannarsa Gianfranco , Corradini Claudio , Nicoletti Isabella , Pizzoferrato Laura , Vivanti Vittorio
Published: May 2005

The electrophoresis of ε‐N‐2‐furoylmethyl‐L‐lysine (furosine) was studied in an attempt to develop a method for the identification and quantitation of this compound in processed food. The effect of pH and composition of electrolyte solution on both the electrophoretic migration of furosine and the electroosmotic flow in a bare fused‐silica capillary of 75 μm internal diameter was investigated. We demonstrate that the addition, to the running electrolyte solution, of N,N,N′,N′‐tetramethyl‐1,3‐butanediamine (TMBD) at concentrations ranging from 20 to 80 mM improves peak efficiency and can be used to modulate the migration time of furosine by controlling the electroosmotic flow which is reversed from cathodic to anodic. In a sample of dried milk subjected to a long period of storage under controlled conditions, furosine could be efficiently and reproducibly separated and quantitated by employing as the running electrolyte 60 mM TMBD titrated to pH 2.5. Capillary electrophoresis is a promising technique for the rapid identification and quantitation of furosine in processed food.
Ultrafast high resolution separation of large DNA fragments by pulsed‐field capillary electrophoresis
Authors: Kim Yongseong , Morris Michael D.
Published: May 2005

Pulsed‐field capillary electrophoresis (PFCE) in buffers containing ultradilute polymer solutions in used to separate long chain dsDNA in less than 4 min. Separations are shown to work with chain lengths below 10 kbp and greater than 1.5 Mbp. Several pulse protocols have been examined. If running time is to be minimized, a field inversion with higher peak amplitude in the forward direction than in the reverse, but with equal pulse durations, provides the best resolution. Other protocols can provide higher resolution, but only with longer resolution. Other protocols can provide higher resolution, but only with longer running times.
Preparative isoelectric focusing and Joule effect: A purification cell that contains a heat exchanger
Authors: Araque Arturo , Jaugey Jean , Javet Philippe
Published: May 2005

Reproducibility in protein purification by preparative isoelectric focusing depends greatly on temperature control during the separation process. A preparative apparatus is described, including a heat exchanger between compartments with isoelectric membranes. The selectivity of the isoelectric membranes was optimized as a function of isoelectric points of the separated proteins. At 2500 V and 60 W, 0.3 g of horse heart myoglobin from 0.2 g of whale skeletal muscle myoglobin could be separated in 1 h. At a total load of 2 g protein, 97% of bovine hemoglobin (2% initial concentration) was purified from bovine serum albumin (0.15%).
Biotinylated trypsin and its application as a sensitive, versatile probe for the detection and characterisation of an ovine chondrocyte serine proteinase inhibitor using Western blotting
Authors: Rodgers Kenneth John , Melrose James , Ghosh Peter
Published: May 2005

Biotinylated trypsin (bT) was used as a probe on Western blots of 10–20% polyacrylamide gradient Tris‐Tricine gels for the detection of serine proteinase inhibitors (SPIs) isolated from extracts of ovine articular cartilage and from chondrocyte conditioned culture medium. The major cartilage SPI, a 58 kDa glycoprotein, was purified by sequential Sephacryl S–300 gel permeation, concanavalin A affinity, anion exchange and Superose 12 fast protein liquid chromatography (FPLC). A low molecular weight SPI of ∼ 6 kDa was also detectable in cartilage extracts after prolonged storage at 4°C and following affinity chromatography on immobilised chymotrypsin, suggesting that the 6 kDa inhibitor may have arisen from proteolytic modification of the 58 kDa SPI. Analysis of chondrocyte conditioned culture medium using the bT detection system revealed that the major SPI present was the 6 kDa species although a small amount of the 58 kDa SPI was also detectable. The large molecular weight ‘native’ cartilage SPI was distinct from ovine α1‐proteinase inhibitor (α1‐PI) when examined by native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing (IEF). The 6 kDa cartilage SPI was indistinguishable from basic pancreatic trypsin inhibitor (BPTI, aprotinin) following sodium dodecyl sulfate (SDS)‐PAGE under reduced and nonreduced conditions. Amino terminal sequencing of the 6 kDa cartilage SPI revealed a strong (90%) homology with BPTL.
Distinction between genotypes of Lupinus species by sodium dodecyl sulphate‐gel electrophoresis and by capillary electrophoresis
Authors: Pollard Naomi J. , Wrigley Colin W. , Bekes Ferenc , Aumatell Anthony , Macritchie Finlay
Published: May 2005

Effective distinction was achieved among a wide range of lupin grain samples by either sodium dodecyl sulphate (SDS)‐gel electrophoresis or capillary electrophoresis, based on grain‐protein composition. Capillary electrophoresis was faster (< 1 h) and provided slightly greater distinction between the samples. On the other hand, SDS‐gel electrophoresis could provide a greater throughput of samples in a 24 h period. Either technique could be used successfully to distinguish between lupin species and cultivars for taxonomic analysis or seed identification.
Capillary electrophoresis and electrochemical detection of underivatized oligo‐ and polysaccharides with surfactant‐controlled electroosmotic flow
Authors: Zhou Weihong , Baldwin Richard P.
Published: May 2005

Complex polysaccharide mixtures were analyzed directly without derivatization by capillary zone electrophoresis in strongly alkaline solutions and electrochemical detection at a Cu electrode. The positively charged surfactant cetyltrimethylammonium bromide was included in the electrophoresis medium in order to reverse the electroosmotic flow and permit elution to be in order of increasing polysaccharide size. Carbohydrate samples analyzed by this approach included linear maltoses, enzymatically hydrolyzed starch, and commercially available dextrans of up to an average molecular weight of 18 300. Detection by constant‐potential oxidation at a Cu electrode was very sensitive, with detection limits for individual carbohydrates generally below the femtomole level.
Indirect UV detection of carbohydrates in capillary zone electrophoresis by using tryptophan as a marker
Authors: Lu Bing , Westerlund Douglas
Published: May 2005

A rapid and sensitive method for the analysis of sugars was developed by capillary zone electrophoresis (CZE) with indirect UV detection. A mixture of nine mono‐ and oligosaccharides was separated in about 20 minutes on a fused silica capillary in a highly alkaline (pH > 12) background electrolyte (BGE). With tryptophan as the marker detected at 280 nm the femtomole level detection of sugars was possible. The system was optimized by studies on the buffer pH, the marker concentration, as well as the inner diameter of the capillary. The indirect detection of sugars was also performed with a cationic marker, N‐benzylcinchonidium chloride (BCDC). However, the detection sensitivity was much lower in comparison to the tryptophan marker system.
Analysis of macrolide antibiotics by capillary electrophoresis
Authors: Flurer Cheryl L.
Published: May 2005

Capillary electrophoresis was utilized in the study of the macrolide antibiotics (i.e. pharmaceutical glycoconjugates) clarithromycin, erythromycin, oleandomycin, troleandomycin, and spiramycin. In order to assist in analyte solubilization, two buffer systems using acetonitrile were developed. The first system involved 30 mM sodium cholate and 20% acetonitrile in 80 mM sodium phosphate, pH 6. This buffer permitted the baseline resolution of all five glycoconjugated antibiotics. In addition, erythromycin was separated from its derivatives estolate and ethylsuccinate. In the absence of surfactants, a higher acetonitrile quantity, 65%, was used in the second buffer system, with 35 mM sodium phosphate, pH 6. Selectivity between oleandomycin and clarithromycin was reversed in this system compared to the cholate buffer, indicating solute interaction with the cholate micelles in the previous system. Calibration linearity and detection sensitivity were improved in the high acetonitrile buffer, due to decreased background absorbance. It was demonstrated that both buffer systems can be utilized for the visualization of minor components that may be present in bulk pharmaceuticals.
Two‐dimensional gel electrophoresis of Escherichia coli homogenates: The Escherichia coli SWISS‐2DPAGE database
Authors: Pasquali Christian , Frutiger Séverine , Wilkins Marc R. , Hughes Graham J. , Appel Ron D. , Bairoch Amos , Schaller Dominique , Sanchez Jean‐Charles , Hochstrasser Denis F.
Published: May 2005

Numerous Escherichia coli proteins have already been characterized by two‐dimensional gel electrophoresis (2‐D PAGE), using carrier ampholytes in the first dimension (VanBogelen, R. A., Sankar, P., Clark, R. L., Bogan, J. A. and Neidhardt, F. C., Electrophoresis 1992, 13, 1014–1054). We present here a reference protein map of E. coli obtained with immobilized pH gradients (IPG) and available in a SWISS‐2DPAGE format. Out of the protein spots identified in the E. coli gene protein database by Neidhardt's group, 153 have been identified on the E.coli SWISS‐2DPAGE database map by gel comparison and most of them were confirmed either by the analysis of amino acid composition (AAC) and/or N‐terminal microsequencing. Additionally, five as yet unsequenced proteins were found. The E. coli SWISS‐2DPAGE database is part of the ExPASy molecular biology server accessible through the Word Wide Web network.
Identification of human myocardial proteins separated by two‐dimensional electrophoresis with matrix‐assisted laser desorption/ionization mass spectrometry
Authors: Thiede Bernd , Otto Albrecht , Zimny‐Arndt Ursula , Müller Eva‐Christina , Jungblut Peter
Published: May 2005

Disease‐associated proteins separated by two‐dimensional electrophoresis (2‐DE) are often in the femtomole range. Identification of 2‐DE separated proteins by sequencing and amino acid analysis is limited to the lower picomole range. Identification down to the femtomole range can be achieved by matrix‐assisted laser desorption ionization‐mass spectrometry (MALDI‐MS). We optimized the measurement by MALDI‐MS for the analysis of proteolytic digests of 2‐DE‐separated proteins. The direct analysis of peptide mixtures can be used for rapid and sensitive protein identification. In some cases, more information about the protein can be obtained by separating the peptides by micro high‐performance liquid chromatography (HPLC) before employing MALDI‐MS analysis. More peptides are found than in the mixture, and comparison of HPLC patterns can reveal some differences to be post‐translational modifications of proteins, even in the case of identical peptide mass fingerprints. Furthermore, carboxy‐terminal sequencing by on‐target carboxypeptidase P digestion can be used to confirm the obtained result without the need for more material. The search program FRAGFIT was modified and renamed FRAGMOD to include the modifications of methionine and tryptophan oxidation and alkylation of cysteine by acrylamide into the mass search. By applying this procedure, 15 proteins were identified, among them two different putative phosphorylated forms of two proteins, a putative N‐terminal blocking group and four dilated cardiomyopathy‐associated proteins. The resulting approach for the identification may be used for large‐scale investigations of 2‐DE‐separated proteins.
The effects of polymer properties on DNA separations by capillary electrophoresis in uncross‐linked polymer solutions
Authors: Barron Annelise E. , Sunada Wade M. , Blanch Harvey W.
Published: May 2005

Low‐viscosity, aqueous solutions of hydrophilic linear polymers have been shown to be useful for the separation of DNA restriction fragments by capillary electrophoresis (CE). However, the choice of polymer type, size, and concentration remains largely empirical, because the mechanism of high‐field electrophoretic DNA separations in polymer solutions is not well understood. To assist in elucidating the mechanism of DNA separation, we experimentally investigated the effects of polymer properties such as stiffness (persistence length), average molecular mass, polydispersity, and hydrophilicity on the separation of DNA ranging from 72 bp to 23 kbp. This was accomplished by comparing the results of DNA separations obtained by counter‐migration CE in dilute and semidilute solutions of linear polyacrylamide (PAA), hydroxyethylcellulose (HEC), and hydroxypropylcellulose (HPC) polymers of several different average molecular masses.
Direct isolation of proteins from sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and analysis by electrospray‐ionization mass spectrometry
Authors: Schuhmacher Martina , Glocker Michael O. , Wunderlin Markus , Przybylski Michael
Published: May 2005

A new method for the isolation of proteins separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), using electroelution with a modified buffer system, is described. The method is suitable for direct characterization by electrospray‐ionization mass spectrometry (ESI‐MS), or alternatively matrix‐assisted laser desorption‐ionization (MALDI), no additional purification steps being required. Following separation by SDS‐PAGE, proteins were stained with Coomassie Blue, and isolated by electroelution using SDS‐free ammonium acetate (pH 2.5) as elution buffer. The polarity of the electroelution system was reversed, which, upon in situ dissociation of protein‐SDS complexes, resulted in migration of free proteins to the cathode under the conditions employed. Recovery rates of 25–58% were determined for model proteins. Analyses by ESI‐MS provided exact molecular weight determinations of isolated proteins and of protein mixtures not resolved by SDS‐PAGE. Essentially SDS‐free molecular ions were obtained, except for bovine serum albumin with one SDS‐adduct. Charge distribution of molecular ions were similar to those of the native proteins. The effects of β‐mercaptoethanol (β‐ME) and dithiothreitol (DTT) during electrophoresis were studied with hen egg white lysozyme, revealing the formation of mixed disulfide adducts between proteins and reducing agents. In a first bioanalytical application, hemofiltrate proteins from a patient with uremia were separated by SDS‐PAGE. An ESI‐MS analysis of the proteins isolated from the two most intensive gel bands enabled exact molecular weight determinations, and demonstrated that a gel band of ca. 17 kDa consisted of two different proteins.
Mass spectrometric characterization of glycosylated interferon‐γ variants separated by gel electrophoresis
Authors: Mørtz Ejvind , Sareneva Timo , Haebel Sophie , Julkunen Ilkka , Roepstorff Peter
Published: May 2005

Glycosylated proteins in polyacrylamide gels were characterized by matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) and glycosidase digestion. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of natural, human interferon‐γ (IFN‐γ) showed two glycosylated variants with apparent molecular masses of 20 and 24 kDa. MALDI‐MS of the intact IFN‐γ, electroeluted from the two bands, confirmed that these correspond to IFN‐γ molecules glycosylated at one or both of the two potential glycosylation sites, respectively. The peptide map obtained by MALDI‐MS after digestion in the gel covers 92% of the IFN‐γ sequence and revealed an N‐terminal pyroglutamate residue and one oxidized methionine residue. One glycosylated peptide was detected after treatment of the peptide mixture with neuraminidase, and the carbohydrate structure partially elucidated by sequential glycosidase digestion monitored by MALDI‐MS. A second glycosylated peptide, due to a very heterogeneous glycan structure, could only be observed after separation of the peptides by high performance liquid chromatography (HPLC).
Field and pore size dependence of the electrophoretic mobility of DNA: A combination of fluorescence recovery after photobleaching and electric birefringence measurements
Authors: Tinland Bernard , Pernodet Nadine , Weill Gilbert
Published: May 2005

By combining an electrophoretic cell with a setup of fluorescence recovery after photobleaching (FRAP) we can measure the electrophoretic mobility μ of double‐stranded lambda DNA in agarose gel as a function of electric field E and gel concentration C. Mobility varies linearly with the field in agreement with the biased reptation model with fluctuations. The slopes are analyzed in term of orientation and compared with birefringence results. The mobility extrapolated at zero field follows the prediction of the reptation theory; we deduced the variation of the pore size with the agarose concentration. With a special use of our setup, we measure directly the free‐mobility μ0 of the DNA.
Mechanisms of retardation of rigid spherical particles with 3 to 1,085 nm radius in capillary electrophoresis, using buffered polyacrylamide (molecular weight 5 × 106) solutions
Authors: Radko Sergey P. , Chrambach Andreas
Published: May 2005

Subjecting particles in the size range of 3 to 1085 nm radius (R) to capillary electrophoresis in buffered solution of entangled uncrosslinked polyacrylamide (Mr 5 × 106), it was found that particle size‐dependent retardation (“molecular sieving”) becomes electric field‐ and particle size range‐dependent once the particle size exceeds 15–20 nm in radius. The field strength dependence of the retardation coefficient [KR = d(log mobility)/d(polymer concentration] and the positive or negative sign of dKR/dR suggest the existence of two different mechanisms of molecular sieving depending on the particle size range: particles with diameters less than the screening length (or blob size) of the polymer network are thought to penetrate into the available spaces within a discontinuous polymer network; particles with diameters larger than the screening length (or blob size) of the polymer network are thought to undergo size‐dependent retardation by exerting shear stress against polymer chains, and displacing them, so as to cause local deformations in a continuous polymer network. A limit in the separating capacity of molecular sieving, due to a sharp increase in the rate of band widening with polymer concentration, was found when the value of the retardation coefficient exceeded 60 (mL/g).
Influence of transference number on migration and deformation of concentration boundaries in capillary zone electrophoresis
Authors: Štědrý Milan , Popelka Štěpán , Gaš Bohuslav , Kenndler Ernst
Published: May 2005

The change of the position and shape of a concentration boundary existing in the background electrolyte in capillary zone electrophoresis is teated theoretically. Such boundaries may occur when the ionic concentration, c, of the sample differs from that of the background electrolyte. Both mathematical treatment and numerical simulation lead to the result that when an (initially symmetrical) concentration boundary migrates in the capillary it becomes nonsymmetrical. The reason for this is the nonsymmetry of the function, which is obtained as the difference of the transference number (which is a function depending on c) and its linear approximation at the average value of the concentration. Numerical simulations for capillary zone electrophoresis with LiI or potassium benzorate as a hypothetical background electrolyte show the formation of asymmetrical plugs for samples of more diluted electrolyte solutions.
Flowable networks as DNA sequencing media in capillary columns
Authors: Menchen Steve , Johnson Ben , Winnik Mitchell A. , Xu Bai
Published: May 2005

A novel class of materials that self‐assemble in water into equilibrium network structures with a well‐defined mesh size consist of polyethylene glycols (PEG's) end‐capped with micelle‐forming fluorocarbon tails. These micellar systems form flowable aqueous gel‐like networks that permit electrophoretic DNA sequencing in capillary columns. The gels have unusual rheological properties, including netword breakdown under shear, resulting in plug flow that allows colums refill with complete ejection of byproducts of the previous sequencing analysis. In this system, DNA fragment electrophoretic mobilities are unaffected by the hydrophobicity of the polymer tails. Low molecular weight (M) PEG chains (M 8000) show catastrophic resolution loss for DNA fragments larger than 100 bases due to band broadening. For a longer PEG segment (M 35000) separating the end groups, band broadening occurs for DNA fragments larger than 300 bases, implying that the PEG segment length controls the mesh size in the equilibrium network structure. Optimum sequencing results were obtained from a 6% solution of a 1:1 mixture of C6F13 end‐capped‐ and C8F17 end‐capped PEG 35000. The resolution limit of fluorescent‐dye‐labeled sequencing products in this formulation was 450 bases in 75 μm capillaries at 200 V/cm.
Characteristics of single‐stranded DNA separation by capillary gel electrophoresis
Authors: Kamahori Masao , Kambara Hideki
Published: May 2005

The optimization of electrophoretic conditions for fast separation of singlestranded DNA by capillary gel electrophoresis was investigated. Mobilities and band broadening of the DNA fragments were measured for a 4%T, 5%C gel at 100–300 V/cm. The resolution of DNA peaks was evaluated for band spacing and band width. Band spacing decreased with increasing electric field strengths as well as DNA fragment size; resolution for large DNA fragments also decreased at higher electric field strengths. The measured mobilities in a 4%T, 5%C gel were analyzed by the repration theory and compared with those in a 9%T, 0%C matrix. The reptation plots indicate that the onset of reptation in a 4%T, 5%C gel occurs at a DNA size of about 200 bases and about 150 bases in a 9%T, 0%C matrix. The 4%T, 5%C gel proved superior to the 9%T, 0%C matrix with respect to resolution and analysis time. The optimum electric field strength and migration distance for specific DNA fragment length (200, 400 and 700 bases) in the shortest time were calculated from band spacing and the measured band width for the 4%T, 5%C gel at Rs = 0.5. Under optimized conditions, analysis of DNA fragments up to 200, 400 and 700 bases could be carried out in about 5, 50, and 230 min, respectively.
Negative staining with zinc‐imidazole of gel electrophoresis‐separated nucleic acids
Authors: Hardy Eugenio , Pupo Elder , Casalvilla Racmar , Sosa Angela E. , Trujillo Luis E. , López Eduardo , Castellanos‐Serra Lila
Published: May 2005

Zinc and imidazole salts were applied for the detection of nucleic acids on either polyacrylamide or agarose gels. After electrophoresis, polyacrylamide gels are washed in distilled water to remove most of the residual electrophoresis reagents, then incubated in 10 mM zinc sulfate for 10 min, and subsequently immersed in 0.2 M imidazole for 3 min. As a result, zinc salts precipitate on the gel surface, except in the positions occupied by nucleic acids, which appear as transparent, colorless bands. Staining of nucleic acids on agarose gels can be performed by incubation in 40 mM zinc sulfate for 10 min, followed by immersion in 0.2 M imidazole for 5 min to form a deep white‐stained background. On soaking in 2 M imidazole for 45 min, the imidazole‐induced zinc precipitate is removed from the positions where nucleic acids are located resulting in a negative image of colorless and transparent nucleic acid bands against a white background. The sensitivity of this stain ranges from 5 to 7 ng/band for small (from 1 to 0.2 kbp) DNA, from 7.8 to 13 ng/ band for different 22‐base oligonucleotides, from 62 to 125 ng/band for large (from 20 to 2 kbp) DNA, and is 1 μg/band for human peripheral‐blood monocyte RNA. After chelation of zinc with EDTA, the nucleic acids can be quantitatively recovered from the gel. The principal advantage of this technique over ethidium bromide staining is evident for preparative purposes. Using zinc‐imidazole in the detection of purified pBACIB.1 (2.8 kbp) plasmid DNA and antiHBsAg single chain Fv antibody fragment (0.7 kbp) DNA, followed by elution from gel slices, ligation and transformation of competent E. coli XL‐1 Blue cells, the number of transformants notably increased from 280 (obtained with conventional ethidium bromide staining plus UV‐irradiation at 312 nm) to 10000.
Direct determination of glutathione in human blood by micellar electrokinetic chromatography: Simultaneous determination of lipoamide and lipoic acid
Authors: Pañak Karina C. , Ruiz Oscar A. , Giorgieri Sergio A. , Díaz Luis E.
Published: May 2005

A reproducible, rapid procedure for the determination of glutathione in human blood by micellar electrokinetic chromatography has been developed. Whole blood samples were deproteinized with 5% w/v sulfosalicylic acid (final concentration). After centrifugation, the supernatant was directly injected for analysis, without further derivatization. Separations were performed by using an uncoated capillary of 30 cm effective length and 50 μm internal diameter (ID), 50 mM Tris‐HCl, 30 mM sodium dodecyl sulfate (SDS), pH 7.00, as running buffer, and 10–20 kV. On‐line detection was carried out at 214 nm and a detection limit in the range of femtomoles was achieved. Under the same experimental conditions, we resolved a mixed standard solution containing glutathione in its oxidized and reduced forms, lipoamide and α‐lipoic acid. The corresponding migration times were reproducible. The present method allows rapid determination of these compounds, which play a critical role in oxidative stress, in cellular defense against injurious agents and whose levels are related to the toxicology and metabolism of several toxins and drugs, such as antineoplastic agents.
Identification of human myocardial proteins separated by two‐dimensional electrophoresis using an effective sample preparation for mass spectrometry
Authors: Otto Albrecht , Thiede Bernd , Müller Eva‐Christina , Scheler Christian , Wittmann‐Liebold Brigitte , Jungblut Peter
Published: May 2005

Peptide mass fingerprinting is a powerful tool for the identification of proteins separated by two‐dimensional gel electrophoresis (2‐DE). The identification of in‐gel digested proteins by peptide mass fingerprinting was significantly improved in comparison to blot‐digests by using a peptide‐collecting device. This device allows the effective purification and concentration of enzymatic digests of low‐intensity spots without expensive equipment and is described in detail. Sensitivity in the fmol range was demonstrated by unequivocal identification of bovine serum albumin after sodium dodecyl sulfate — polyacrylamide gel electrophoresis. Furthermore the high performance liquid chromatography pattern of in‐gel digests indicated a 2‐ to 3‐fold higher yield of the separated peptides. Therefore, a higher amount of the peptides was available to perform N‐terminal sequencing. The identification of 16 proteins from a high‐resolution 2‐DE gel map of human myocardium tissue has been achieved by means of this technique. Three of these proteins were associated with changes in spot intensity with dilated cardiomyopathy.
Efficiency of separation of DNA mutations by constant denaturant capillary electrophoresis is controlled by the kinetics of DNA melting equilibrium
Authors: Khrapko Konstantin , Coller Hilary , Thilly William
Published: May 2005

Constant denaturant capillary electrophoresis (CDCE) separation takes place in the heated portion of the capillary where faster‐moving, unmelted DNA fragments are in equilibrium with slower‐moving, partially melted forms. Within a certain temperature range, the position of the melting equilibrium and thus the average electrophoretic mobility of each mutant is different. The resulting difference in mobility allow sequences containing single base pair point mutations to be separated from each other. We report the results of experiments in which we explored the rules defining separation efficiency by varying the parameters of CDCE. We discovered an unusual peak broadening mechanism. In contrast to most other DNA electrophoresis systems, peak width in CDCE steadily decreases with the square root of the separation speed. Moreover, the peak width displays a sharp maximum at a specific temperature. To account for these observations, we use a model which describes CDCE separation as a random walk. According to this model, peaks in CDCE are broad because the kinetics of the melting equilibrium are slow and there‐fore the number of random walk steps represented by melting/renaturation transitions is relatively small. In addition to providing a satisfactory interpretation of the data, the model also predicts that separation efficiency will increase as the ionic strength of the running buffer is increased and as the concentration of denaturant in the buffer is decreased. These predictions were verified and were used to establish conditions for high‐resolution CDCE suitable for separating complex mixtures of single base pair mutants.
Linearity evaluation in absorbance detection: The use of light‐emitting diodes for on‐capillary detection in capillary electrophoresis
Authors: Macka Miroslav , Andersson Per , Haddad Paul R.
Published: May 2005

A model which takes into account both stray light and polychromatic light was used to predict and evaluate linearity in on‐capillary detection in capillary electrophoresis (CE). According to the model the stray light is the major factor which determines linearity under typical CE operating conditions. By calculating theoretical absorbance versus concentration plots, the influence of different levels of stray light and polychromatic light on linearity is demonstrated. Experimentally, six light‐emitting diodes (LEDs) in the range from 563 to 654 nm were examined as light sources for on‐capillary detection in CE. Fitting theoretical curves to measured linearity plots enabled determination of the values of both effective path lenght and stray light for a particular detection system. The detector linearity for the four LEDs was compared to mercury and tungsten lamps used with interference filters. For potassium permanganate as the test compound, the linear range for a 563 nm LED was two times greater than that for a mercury lamp operated at 546 nm. The relatively poor linearity of the mercury lamp detector is explained by its high level of stray light. The noise of the LED563‐based detector was the same as for the mercury lamp, whereas the other LEDs of higher light intensity gave approximately half the noise of the mercury lamp. The lowest noise level of 3 × 10−5 AU was obtained for the LED at 554 nm (determined at a detector time constant of 0.1 s).
A practical procedure for the determination of association constants of the analyte‐chiral selector equilibria by capilllary zone electrophoresis
Authors: Fanali Salvatore , Boček Petr
Published: May 2005

A practical procedure is proposed for the determination of association constants and mobility of the associate of a solute with a chiral selector in chiral separations by capillary zone electrophoresis. The procedure is based on the measurement of the effective mobility of a solute at zero and two different nonzero concentrations of the chiral selector. Simple explicit formulas have been derived in order to calculate the required data. The essence of the procedure is that all mobility data are adjusted to the background electrolyte (BGE) without chiral selector, serving as the viscosity reference. A simple procedure is described for measuring the viscosity of the operational electrolytes directly with the commercial capillary electrophoresis instrumentation, and Walden's rule has been utilized for adjusting the experimental mobility data to constant reference viscosity. The use of the procedure is exemplified by a separation of D,L‐tryptophan in BGE containing α‐cyclodextrin as chiral selector.
Enantioseparation of amino acids and dipeptides using vancomycin as chiral selector in capillary electrophoresis
Authors: Wan Hong , Blomberg Lars G.
Published: May 2005

Vancomycin was applied as chiral selector for the enantiomeric separation of derivatized amino acids and dipeptides. The influence of vancomycin concentration, pH and presence of 2‐propanol in the buffer were examined in order to find optimal separation conditions. Optimization was by factorial design. Further, chiral separation of derivatives prepared with three different reagents was compared. These reagents were 9‐fluorenylmethyl chloroformate (FMOC), 2‐(9‐anthryl)ethyl chloroformate (AEOC) and dansyl chloride (dansyl). Optimum resolution was at high vancomycin concentrations, while optimum efficiency was at low vancomycin concentrations. As a consequence of the very high enantioselectivity of vancomycin, the vancomycin concentration below the amount necessary for maximal resolution can be used. Separation efficiency was relatively low, and this could be attributed to adsorption of the selector at the capillary wall. Three factors led to decreased adsorption: application of a pH above the zero mobility pH value, low vancomycin concentrations and the presence of 2‐propanol. For amino acids, the resolutions of the different derivatives were: dansyl > AEOC > FMOC, while for dipeptides, the highest selectivity was with AEOC.
Characterization of the stereoselective metabolism of methadone and its primary metabolite via cyclodextrin capillary electrophoretic determination of their urinary enantiomers
Authors: Lanz Matthias , Thormann Wolfgang
Published: May 2005

Using capillary zone electrophoresis with a phosphate buffer at pH 3, containing 4.3 nM hydroxypropyl‐β‐cyclodextrin as chiral selector, the simultaneous separation of the enantiomers of methadone and its primary metabolite, 2‐ethylidene‐1,5‐dimethyl‐3,3‐diphenylpyrrolidine (EDDP), is reported. After solid‐phase extraction and analysis of the reconstituted extracts in a 60 cm fused‐silica capillary of 50 μm ID within about 25 min, the mean S/R enantiomeric ratio of methadone in the urines of eight patients undergoing methadone therapy is shown to be 0.653 (range: 0.502–0.842). The mean enantiomeric ratio for the metabolite is 0.630 (range: 0.517–0.729). These data document the stereoselectivity of the methadone metabolism. Finally, the EDDP/methadone ratio is shown to vary between 0.357 and 2.917 with a mean value of 1.731. The capillary electrophoretic assay described is simple, inexpensive and neither requires any sample derivatization, nor large amounts of organic solvents or expensive separation columns.
Pore size of agarose gels by atomic force microscopy
Authors: Pernodet Nadine , Maaloum Mounir , Tinland Bernard
Published: May 2005

The pore size of agarose gel in water at different concentrations was directly measured using atomic force microscopy (AFM). The experiment was specially designed to work under aqueous conditions and allows direct observation of the “unperturbed” gel without invasive treatment. The pore size a as a function of gel concentration C shows a power law dependence a ˜ C−γ, where γ lies between the prediction of the Ogston model for a random array of straight chains, 0.5, and the value predicted by De Gennes for a network of flexible chains, 0.75. We confirm that gels present a wide pore size distribution and show that it narrows as the concentration increases.
Application of two‐dimensional gel electrophoresis to Prunus armeniaca leaf and bark tissues
Authors: Faurobert Mireille
Published: May 2005

The protein composition of Prunus armeniaca bark and leaf tissues was investigated by two‐dimensional gel electrophoresis. Three different extraction procedures were tested in order to obtain reproducible gels with numerous spots of high intensity. The best results were achieved with extraction in Tris‐buffer in the presence of a nonionic detergent, reducing agents, and polyphenol oxidase inhibitors. As many as 744 protein spots were resolved from leaf tissues. The patterns exhibited well‐focused spots, with apparent molecular masses ranging from 19 to 90 kDa and isoelectric point from 4.5 to 8.5. The Tris extraction buffer was also the most appropriate for cortical tissue analysis.
Analysis of domoic acid isomers in seafood by capillary electrophoresis
Authors: Zhao Jian‐Ying , Thibault Pierre , Quilliam Michael A.
Published: May 2005

Methods for the analysis of domoic acid (DA) based upon capillary electrophoresis (CE) combined with UV absorbance detection were investigated. DA could be analyzed using bare fused‐silica capillaries in either the cationic or anionic mode with acidic or basic buffer systems, respectively. Highest performance, in terms of both separation efficiency and analysis time, was achieved with phosphate or borate buffers at a pH of approximately 9. The addition of (3‐cyclodextrin to the borate buffer permitted a separation of DA and several of its isomers (isodomoic acids) that was superior to that achieved with liquid chromatography (LC). The optimum background electrolyte for the separation was 22.5 mM sodium tetraborate at pH 9.2 with 20 mM (3‐cyclodextrin. In addition, an extraction and clean‐up procedure was developed and tested with mussels, clams and anchovies. Aqueous methanol extraction of samples followed by a tandem strong anion and strong cation exchange clean‐up provided an extract that was completely compatible with CE analysis. A mass detection limit of 3 pg of DA injected and a method detection limit of 150 ng/g in tissues could be achieved. Comparison with LC showed that comparable precision and accuracy could be attained by the two techniques.
Reproducibility of polypeptide spot positions in two‐dimensional gels run using carrier ampholytes in the isoelectric focusing dimension
Authors: Lopez Mary F. , Patton Wayne F.
Published: May 2005

The reproducibility of complex protein patterns in two‐dimensional (2‐D) gels run with carrier ampholytes in the first dimension has been investigated. Two different laboratories collaborated in the study and 18 or 19 gels were run in each laboratory for comparison. The electrophoresis chemicals, running devices, and samples were standardized in both labs. The resulting 37 gels were scanned with a charge‐coupled device (CCD) camera and spots were located, counted, quantified, and matched using a commercially available image analysis system. Subsequently, the reproducibility of spot position was determined. To perform the statistical analysis, the test gels were initially each matched to a master references gel. Next, three sets of 12 gels (the image analysis software database could analyze only 12 gels at a time) were analyzed and the isoelectric point (pI) and molecular weight (Mr) positional variation of all the spots that matched across the gels in each set was determined. The resulting statistical analysis indicates very high reproducibility of the carrier ampholyte technique.