bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective influencing of the migration time of SO42− by forming neutral species with Pb2+ in capillary zone electrophoresis
Authors: Groh Thomas , Bächmann Knut
Published: May 2005

Organic and inorganic anions are analyzed using capillary zone electrophoresis with indirect fluorescence detection. As electrolyte 2,5‐dihydroxybenzoic acid is used. By adding Pb2+ to the elctrolyte the migration time of SO42− can be selectively influenced and the separation of C1− and SO42− considerably improved. The fluorescence intensity of 2,5‐dihydroxybenzoic acid increases in the presence of Pb2+ up to a concentration of 6*10−4 mol/L Pb2+. Limits of detection in the lower μmol/L range were obtained.
DNA digest analysis with capillary electrophoresis
Authors: Paulus Aran , Hüsken Dieter
Published: May 2005

DNA digest analysis with polymer‐filled capillaries in capillary electrophoresis is described. The samples analyzed consisted of commercially available standards including a 100 base pair ladder with repeating units up to 2000 base pairs. Three DNA digests covered the most common small fragment ranges: up to 600 base pairs, up to 1500 base pairs and from 100 to 2500 base pairs. All samples were separated by traditional slab gel electrophoresis at various agarose concentrations and by automated capillary electrophoresis. The capillary electrophoretic separations were achieved with noncross‐linked polyacrylamide from 6% monomer solutions. The acrylamide was polymerized inside the capillary, which was coated with a methacryloxysilane to insure binding of the polyacrylamide to the capillary wall. With 6% columns, excellent separations were observed up to 600 base pairs with base line resolution for fragments differing in less than 10 base pairs. Resolution power for fragments between 600 and 2200 decreased to about 300 base pairs. Compared to slab gels, capillary electrophoresis achieved better resolution in the low fragment range, whereas with the reported column composition, slab gels were superior above 600 base pairs. Fast access to the analysis of a specific sample, automation, and a larger dynamic range for sample load are further benefits of a DNA digest analysis by capillary electrophoresis.
The silver staining procedure of sodium dodecyl sulfate‐gels may be accelerated by shortening fixation time
Authors: Kirkeby Svend , Moe Dennis , Bòg‐Hansen Thorkild C.
Published: May 2005

In most silver staining methods the first step in the staining of proteins separated after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis is a rather protracted fixation of the gels. Optimum fixation should be short, cause no background staining and effectively immobilize the proteins in the gel without masking the proteins for reaction with the staining solution. Further, the concentration of the fixing compounds should be as low as possible due to the potentional toxicity of fixatives. Fixation for only 5 min with mixtures of very low concentrations of formaldehyde and glutaraldehyde in ethanol, or a solution of formaldehyde or glutaraldehyde in picric acid and ethanol, fulfill these demands, provided that the gels were prefixed in ethanol‐acetic acid for 10 min. As a consequence of these results a fast and sensitive silver staining procedure is proposed.
Enhancing spot detection and reducing noise from digitized electrophoretic gel images using area processing filters
Authors: Patton Wayne F. , Tempst Paul
Published: May 2005

Twenty area processing filters and filter combinations were evaluated in an effort to optimize presentation of two‐dimensional electrophoretic profiles to the Laplacian spot finder for maximal spot detection sensitivity. Images of electrophoresis gels were obtained by digitizing polyacrylamide gels at 1024 × 1024 picture elements (pixels) resolution with 256 grey scale levels using the charge coupled device (CCD) camera of the Millipore Bio Image 110S computerized imaging system. The images were imported into an Apple Macintosh microcomputer and selectively enhanced by applying various area processing filters. Previously described least squares fit, low‐pass, Gaussian and median filters were used to reduce noise in the digitized images. These filters differ in that during the summation process the least squares template weighs the immediately adjacent pixels more heavily than the Gaussian template. The low‐pass filters weigh all neighboring pixels equally. Median filters replace the pixel of interest with the middle (median) value of the pixel neighborhood. An analysis of convolution filter sizes indicated that a 7 × 7 matrix was optimal for 22 cm × 22 cm gels. When using the median area processing procedure, however, the 3 × 3 filter was found to be superior to the 7 × 7 filter. The 7 × 7 least squares filter significantly improved detection of low abundance polypeptides while having only minimal effects on the high abundance polypeptides. The 7 × 7 Gaussian and 3 × 3 median filters also improved detection of low abundance polypeptides but reduced the integrated areas of the high abundance polypeptides and thus their integrated optical densities as well. The 7 × 7 least squares and the 3 × 3 median filters effectively increased spot detection, and reduced noise with minimal alteration in the modal distribution of spot areas and integrated optical densities in the image. They also preserved image quality (minimal blurring). The practical consequence of using the 7 × 7 least squares, 3 × 3 median or 7 × 7 Gaussian filters is an 8‐fold increase in sensitivity due to noise reduction, resulting in the detection of 60–65% more polypeptides in a typical silver stained gel.
A new concept for separating nucleic acids by electrophoresis in solution using hybrid synthetic end labelled‐nucleic acid molecules
Authors: Noolandi Jaan
Published: May 2005

Analogy between the symmetry breaking of the electrical driving force and the opposing friction force in gels using pulsed electric fields is made with the corresponding effect for polyelectrolyte coils in solution related to the molecular weight‐independent charge density. The synthesis of hybrid molecules to break the symmetry of constant charge density is proposed, in which standard polypeptide end labels are attached to nucleic acid fragments. The combinatorial chemical library scheme of Brenner and Lerner (Proc. Natl. Acad. Sci. USA 1992, 89, 5381–5383), making use of hybrid molecules for another purpose, is mentioned, with the suggestion that for electrophoresis in solution a neutral end‐labelled polypeptide chain would give the largest molecular weight and time‐dependent change in the charge density for pulsed fields.
Detection by Nile Red of agarose gel electrophoresed native and modified low density lipoprotein
Authors: Greenspan Phillip , Gutman Robert L.
Published: May 2005

The use of Nile Red to track and stain low density lipoprotein (LDL) and modified LDL in agarose gels was investigated. Lipoproteins were prestained with Nile Red, a fluorescent dye, prior to electrophoresis. After 2 h of electrophoresis, the LDL and modified LDL were visualized using a UV transilluminator with an excitation wavelength of 302 nm. Spectrofluorometric analysis revealed that the Nile Red fluorescence of the stained LDL had an emission maximum of 609 nm. This rapid staining method of LDL and modified LDL can detect as little as 2.5 μg of LDL protein and permits the immediate visualization of these lipoproteins in agarose gels.
Identification of predominant T‐cell receptor rearrangements by temperature‐gradient gel electrophoresis and automated DNA sequencing
Authors: Segurado Oscar G. , Schendel Dolores J.
Published: May 2005

To assess the diversity of T‐cell receptor (TCR) gene rearrangements in uncloned lymphocytes we used a three‐stage strategy that allows the detection of a restricted TCR repertoire and the identification of the predominant, rearranged sequence(s). We have analyzed in parallel T cells obtained from a renal cell carcinoma infiltrate that specifically lyse the autologous tumor after in vitro culture and T cells from autologous peripheral blood. First, DNA amplification by the polymerase chain reaction (PCR) was performed with a number of oligonucleotide primers specific for several TCR Va gene families. All Vα primers displayed specific amplification products in the peripheral blood, while a restricted TCR repertoire was present in the tumor‐infiltrating lymphocytes. Subsequently, positively amplified PCR products were run in a temperature‐gradient gel electrophoresis. A limited number of bands corresponding to predominant homo‐ and heteroduplexes was only found in the tumor‐infiltrating lymphocytes. The presence of a low number of rearranged TCR sequences in these samples was confirmed by automated single‐stranded DNA sequencing using a single fluorescent dye. These results support the broad application of this strategy for targeted sequencing of those PCR products carrying predominant DNA templates without previous DNA cloning.
Horizontal semi‐dry electroblotting for the detection of the low density lipoprotein receptor in solubilized liver membranes
Authors: Himber Jacques
Published: May 2005

A high efficiency transfer of the low density lipoprotein (LDL) receptor proteins from polyacrylamide slab gel onto immobilizing nitrocellulose membranes using the horizontal semi‐dry electrophoretic system is described. The transfer of the LDL receptors from solubilized rat liver microsomes was performed between two graphite plate electrodes in a continuous buffer system containing methanol and sodium dodecyl sulfate. The protein transfer was achieved in only 150 min at a constant current of 0.8 mA/cm2 at room temperature with very low Joule heat development. The homogeneous electric field yield between the two electrode plates produced a satisfactory transfer of the LDL‐receptor protein band in spite of its high molecular weight, and only few protein traces remained in the polyacrylamide gel after blotting. This improved method allows a rapid and quantitative transfer of the LDL receptors without protein denaturation, since the specific binding activity of the blotted receptor is retained as demonstrated by ligand‐blotting and immuno‐blotting.
A study of the advantages and limitations of immunoblotting procedures for the detection of antibodies against influenza virus
Authors: Kapaklis‐Deliyannis Georgia P. , Drummer Heidi E. , Brown Lorena E. , Tannock Gregory A. , Jackson David C.
Published: May 2005

An immunoblotting procedure was used to determine the specificity and examine some of the properties of antibodies produced following infection of mice with influenza virus or inoculation with noninfectious material with Alhydrogel or complete Freund's adjuvant. The noninfectious material used was β‐propiolactone‐inactivated influenza virus and a preparation (HANA) enriched for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). When influenza viral proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) under nonreducing conditions, each of the anti‐viral antisera tested exhibited strong binding. Under reducing conditions, however, much weaker binding was observed especially towards the HA1 subunit of HA. This was particularly apparent with antisera raised to virus or HANA in the absence of adjuvant. A panel of monoclonal antibodies directed to HA also bound well to viral HA separated by SDS‐PAGE under nonreducing conditions but failed to recognize epitopes on HA1 separated under reducing conditions. These results suggest that when HA is reduced and immobilized on a solid support, it does not display the conformational features essential for the integrity of all epitopes. The immunoblotting procedure was also used to determine the isotype of anti‐viral antibody directed against individual viral proteins and to detect matrix protein 2 (M2) in purified influenza virions and influenza‐infected cells using antisera raised to a synthetic peptide representing a sequence within the M2 protein.
On the use of dimensionless parameters in acid‐base theory. IV. The pH of water solutions of acids, bases, and simple ampholytes
Authors: Rilbe Harry
Published: May 2005

Exact relations between pH and concentrations of water solutions of acids, bases, and simple ampholytes are presented in the form of computer‐created curves. These are mathematically analysed with respect to linearity and inflexion points. The extreme invariance of pH in the immediate vicinity of the isoelectric points of ampholytes is demonstrated in curves of the logarithm of molarity as a function of the logarithm of | pH − pI |. These considerations include a discussion of the suitability of ampholytes as pH standards.
Separation of acidic barley endosperm proteins by two‐dimensional electrophoresis
Authors: Flengsrud Ragnar
Published: May 2005

Proteins in barley aleurone and starchy endosperm from developing seeds were separated by two‐dimensional (2‐D) electrophoresis according to O'Farrell's method. The effect of different extraction methods on the separation was compared. The two best extraction methods were compared by amino acid analysis of extracts and residues. Some tissue‐specific proteins in both tissues are described. Different methods for transfer of separated proteins onto membranes were compared. Amino terminal and internal sequences were determined after transfer of separated proteins or their proteolytic products onto membranes. Amino acid sequence data allowed the identification of 12 different proteins, while 6 remain unidentified and 8 appeared to have a blocked amino terminal.
A short history of electrophoretic methods
Authors: Vesterberg Olof
Published: May 2005

High resolution separation of proteins, based on charge differences, is possible with disc electrophoresis, displacement electrophoresis (isotachophoresis) and notably isoelectric focusing (IEF). Size separation is obtained in sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The combination of gel IEF, followed by SDS‐PAGE in a second‐dimensional slab gel, i.e. two‐dimensional gel electrophoresis, affords the highest resolution with up to several thousand spots per gel. Staining of proteins gives high resolution patterns which can be scanned and stored in comprehensive databases. Over the last 10 years the electrophoretic separation in gels and subsequent visualization of nucleic acids (DNA, RNA) and even genes as well as nucleotides have been much improved, making possible efficient mapping of the genes in humans and all other organisms. This has led to the biggest concerted endeavor in the history of science, i.e. the mapping of the human genome, which will be of importance as long as mankind exists. In the last years electrophoresis in capillaries has attracted much interest because for numerous substances, such as proteins nucleic acids, pharmaceuticals, metabolites, and peptides, it offers high resolution on the analytical scale with over 1 million theoretical plates. Electrophoretic methods have unprecedented impact on life sciences, providing a basis for unique advances in biochemistry, molecular biology, genetics, gene technology and medicine.
The variance and the quantity 2Dt in capillary zone electrophoresis
Authors: Virtanen Rauno
Published: May 2005

The variance is generally used as a measure of dispersion of zones in capillary zone electrophoresis (CZE). It is a quantitative measure of the separation power of a system and different causes of dispersion can be rated by their partial variances, which can be summed up to total variance. However, the additivity is only valid for independent dispersion sources, a fact that often seems to be ignored. The ubiquitous dispersion source diffusion is taken into consideration by the Einstein term 2Dt. Other sources of dispersion are, e.g., injection, detection, thermal gradients, adsorption, and hydrodynamic flow. For each of these sources various variance expressions have been derived. The origin of the term 2Dt and its relation to the variance is explained and the calculation of variance in general is discussed. The equivalence of the diffusion variance and the term 2Dt is verified with some simple initial forms of sample zone and the additivity of variances in ideal zone electrophoresis is demonstrated. The change of conductivity in zones results in asymmetrical zone forms which is an indication of nonideality of a system. It is shown that in such cases the term 2Dt is no longer valid and its use as an additive variance component leads to an erroneous total variance. Because in zone electrophoresis conductivity in a zone always changes more or less, the additivity of variances is never perfectly valid. However, in many cases the nonideality may be so small that the additivity in practice is still applicable. A generally valid way to calculate theroretically the total variance of a zone is to derive a functional representation of the distribution and then calculate the variance from it. This is possible only in the simplest cases. Usually the distribution must be calculated by numerical methods.
Isoelectric focusing in immobilized pH gradients of the glucose transporter from human red cell membranes
Authors: Englund Ann‐Kristin , Lundahl Per
Published: May 2005

Isoelectric focusing of the human red cell glucose transporter (a transmembrane protein) was performed in immobilized pH gradients. Isoelectric focusing of integral membrane proteins presents problems that are related to the amphiphilic nature of these proteins. Solubilizing additives must be used to counteract hydrophobic effects. In our case, urea and the nonionic detergent, Triton X‐100, were used. Focusing was done at 15°C. The isoelectric point (pI) of the glucose transporter (freshly purified by anion‐exchange chromatography in the presence of octyl glucoside) was determined at 8.4 ± 0.05 (n = 9), in good agreement with our earlier determinations by two‐dimensional electrophoresis with isoelectric focusing in the presence of carrier ampholytes in the first dimension. The width of the focused zone was ≈ 0.1 pH unit, more narrow than after focusing with carrier ampholytes. In an immobilized pH gradient from pH 7 to 10, the transporter region at pH 8.4 comprised one major and one or two minor zones. The pH interval 4–10 was also used and showed a single transporter zone. The glucose transporter tends to self‐associate in detergent solution. Octyl glucoside‐purified glucose transporter formed oligomers during incubation at 37°C. Upon focusing, these oligomers appeared in a wide pH interval far below pH 8.4.
Electrophoretic analysis of stimulated cat parotid saliva
Authors: Marshall Tom , Williams Katherine M. , Ekström Jürgen , Tobin Gunnar , Bayard Cecilia , Vesterberg Olof
Published: May 2005

The proteins of parasympathetically stimulated cat parotid saliva were analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and high resolution two‐dimensional electrophoresis (2‐DE). SDS‐PAGE revealed up to 30 polypeptide bands in microliter volumes of unconcentrated saliva. The patterns were highly reproducible and characterized by prominent bands of Mr 57000, Mr30000 and Mr 15000. The major protein (Mr 30000) appeared as a dimer (Mr 60000) when electrophoresed under non‐reducing conditions but dissociated into its monomeric form when the SDS concentration of the denatured samples was increased from 1 to 5%. This indicates a noncovalent association. The protein patterns of saliva from different cats differed slightly but sequential samples from the same cat (collected during 90 min of stimulation) showed little change in protein pattern apart from a fall in total protein content. Following 2‐DE, the major protein (Mr 30000) appeared as a complex array of at least eight spots in two tiers (pI 5.2–6.2; Mr 28000 and 32000). The characteristics of this protein are discussed with reference to allergy to cats.
Micropreparative two‐dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins
Authors: Bjellqvist Bengt , Sanchez Jean‐Charles , Pasquali Christian , Ravier Florence , Paquet Nicole , Frutiger Séverine , Hughes Graham J. , Hochstrasser Denis
Published: May 2005

We describe some simple modifications to the micropreparative two‐dimensional (2‐D) electrophoresis procedure using immobilized pH gradients in the first dimension and sodium dodecyl sulfate‐electrophoresis in the second dimension. The geometry of the immobilized pH gradient strips has been changed to allow the use of large sample application cups that can accomodate greater sample volumes. The use of narrow range immobilized pH gradients with a large sample loading volume allows an efficient resolubilization of polypeptides after the first dimension. As a result, the vertical streaking caused by too high a protein concentration is eliminated in the second dimension. Protein identification by N‐terminal sequencing is facilitated by the large protein load (1–15 mg) which can be employed using this modification. Spots not normally detectable on conventional analytical 2‐D maps, even with sensitive silver staining, are observed. Results for plasma and liver proteins are shown.
Counterflow in isotachophoresis: Computer simulation and experimental studies
Authors: Deshmukh Ranjit R. , Bier Milan
Published: May 2005

A computer model has been developed to predict the effects of co‐ and counter‐current flows on electrophoretic transport. Model predictions are compared with experimental data obtained with the large‐scale recycling isotachophoresis apparatus, recently developed in our laboratory. There were no significant changes in the steady state properties or the transient development of the Kohlrausch‐adjusted profiles with and without flow. The application of counterflow in free flow isotachophoresis in early stages, before a steady state is achieved, can result in selective wash‐out of slower sample components. Thus, the magnitude of counterflow can be used as a variable in optimizing separations. The characteristic features of the model render it also suitable for the simulation of electroosmotic flow in capillary electrophoresis in open tubes, where plug‐type flow prevails.
Clonal imbalances of plasma/serum immunogloblin production in infants
Authors: Tissot Jean‐Daniel , Hohlfeld Patrick , Hochstrasser Denis F. , Tolsa Jean‐François , Calme André , Schneider Philippe
Published: May 2005

To study the clonal events occurring during ontogeny of the humoral immune system, we evaluated plasma immunoglobulin (Ig) production in term newborns and young children by high‐resolution two‐dimensional gel electrophoresis. The clonality pattern of Ig light (L) chains from healthy newborns (n = 19) was similar to that observed on protein maps of their mothers or of normal adults (n > 100), that is, rare distinguishable small spots in a cloud‐like large band of indiscrete Ig L chain spots (polyclonal pattern; maternal Igs). Analysis of plasma samples obtained from infants between 1 month and 5 years of age (n = 55) revealed discrete but evident alterations of the clonality of Ig production. Between the 2nd and 4th months of life, transient attenuation of the “polyclonal background” was observed in association with the appearance of an increasing number of well‐resolved Ig L chain spots (corresponding to plasma Ig concentrations between 0.5 and 1 g/L per spot). This “restricted” clonal pattern was progressivly less apparent on protein maps of infants older than 2 year and evolved towards a “normal” adult polyclonal pattern at the age of 5. These results suggest that the development of the B‐cell clones is heterogeneous, either through limited outgrowth of precursor cells or through selective antigenic pressures.
Comparative mapping of the Pseudomonas aeruginosa PAO genome with rare‐cutter linking clones or two‐dimensional pulsed‐field gel electrophoresis protocols
Authors: Römling Ute , Tümmler Burkhard
Published: May 2005

The Spe1 map of the Pseudomonas aeruginosa PAO (DSM 1707) chromosome was constructed by utilizing two‐dimensional pulsed‐field gel electrophoresis (PFGE) and rare‐cutter linking clones. After end‐labeling and fluorescence staining of macrorestriction fragments had been combined, the two‐dimensional PFGE analyses of partial‐total digests and reciprocal double digests were sufficient for the placement of all fragments on the genomic map. Spe1 linking fragments were isolated from BamH1, Pst1, and EcoR1 genomic libraries of P. aeruginosa PAO. After separation of the heterogeneously sized populations of Spe1‐linearized and uncut circular plasmid DNAs by field inversion polyacrylamide gel electrophoresis, the gel‐eluted linear DNAs were recircularized and subcloned. The 46 analyzed Spe1 linking clones recognized 16 of the 38 fragment links on the Spe1 genome map of P. aeruginosa PAO. The alignment with linking clones was consistent with that obtained from two‐dimensional PFGE mapping protocols.
Progress in developing improved programs for pulsed field agarose gel electrophoresis of DNA
Authors: Arshad Malik F. , Dunn Frederick J. , Vega Raul , Valvano Jonathan W. , Serwer Philip
Published: May 2005

Details are described here for using a rotating gel to perform pulsed field agarose gel electrophoresis (PFGE) with programmable control of the following variables: magnitude of the electrical field, polarity of the electrical field, temperature of the gel and position of the rotating disk upon which the agarose gel rests. By use of this procedure for programmable control, modes of PFGE have been explored that have the following characteristics: (i) resolution by DNA length is completely lost for DNA shorter than a critical length that increases as the pulse times increase, and (ii) resolution by DNA length is enhanced for longer DNAs that are shorter than a second critical length. This window of resolution can be moved to the position of the 2–6 Mb chromosomes of Schizosaccharomyces pombe.
The characterization of snake venoms using capillary electrophoresis in conjunction with electrospray mass spectrometry: Black Mambas
Authors: Perkins John R. , Parker Carol E. , Tomer Kenneth B.
Published: May 2005

Capillary electrophoresis has been used in conjunction with electrospray mass spectrometry using both full‐scan and selected ion monitoring modes to supply as much information as possible about the venom of Dendroaspis polylepis polylepis (Black Mamba). As an example of the application of capillary electrophoresis/electrospray mass spectrometry (CE/ESI/MS) to the analysis of a complex mixture of small proteins, we have analyzed the venom of Dendroaspis polylepis polylepis using the combined techniques. Both full‐scan and selected ion monitoring modes were employed. CE/ESI/MS provides a rapid and extremely sensitive method for molecular weight determination, particularly when selected ion monitoring is employed. It has been utilized to provide sequence confirmation for those toxins which have already been described in the literature. Our methodology indicates the presence of at least 70 peptides in the molecular weight range 6000–9000.
Fast analysis of proteins by isoelectric focusing performed in capillary array detected with concentration gradient imaging system
Authors: Wu Jiaqi , Pawliszyn Janusz
Published: May 2005

Isoelectric focusing of several protein samples is performed simultaneously in an array of 2–4 short capillaries (4–10 cm long). The separated proteins are detected by either an on‐line concentration gradient imaging detection system, or a single point concentration gradient detector which requires a mobilization step following the focusing process. The application of the capillary array increases the throughput of the capillary isoelectric focusing (CIEF) technique, and makes the technique comparable in sample throughput to the gel slab isoelectric focusing technique, but with much faster speed of separation and quantitation. The analysis is completed in 2–3 min with the imaging detection system, which is about 100 times faster than the conventional gel slab isoelectric focusing and 10 times faster compared to conventional CIEF. The resolution of the separation is 0.02 pH units or better. All separated proteins in the capillary array are detected by the universal concentration gradient detector, which eliminates the need for sample derivatization. The concentration limit of detection for the inexpensive instrument described in this work is about 30 μg/mL and can be reduced by using higher quality components in the system, such as a more stable laser, higher optical quality capillaries and a faster computer A/D board. The sample consumption is 400 nL for the 4 cm long, 100 μm ID square capillary. The isoelectric point (pI) values of the samples can be directly determined without internal pI markers from their positions inside the capillary after focusing when the imaging detection system is used.
Ultrasensitive plasmid mapping by high performance capillary electrophoresis
Authors: Maschke Hans E. , Frenz John , Belenkii Alexi , Karger Barry L. , Hancock William S.
Published: May 2005

This paper compares high performance capillary electrophoresis (HPCE) and conventional slab electrophoresis in mapping of four closely related plasmids with three different restriction enzymes. The plasmids express full length and truncated forms of a growth factor receptor oncogene product and were digested with HpaII, HaeIII and RsaI. The resulting oligonucleotide fragments were under 2000 base pairs in length, a size well suited to separation by HPCE with linearpolyacrylamide as a sieving matrix. Plasmid mapping is an essential tool in biotechnology both for the design of an expression system and for monitoring the stability of the expression system during fermentation. HPCE can yield much higher resolution of oligonucleotides than attainable in conventional agarose gel electrophoretic procedures for plasmid mapping. In the examples described here, the HpaII digests provided the surest identification of individual plasmids in the HPCE analysis and could discriminate among all four plasmids. In conventional slab electrophoresis, however, the RsaI digests provided the best discrimination, although two of the plasmids in this system yielded essentially identical electrophoretic patterns. Hence the optimal restriction enzyme for plasmid mapping applications with HPCE may differ from that selected on the basis of conventional slab gel analysis, and the former technique can provide higher discrimination among related plasmids. The advantages of the HPCE format with respect to speed, low sample consumption and resolution are described.
Conventional isoelectric focusing and immobilized pH gradients in ‘macroporous’ polyacrylamide gels
Authors: Wenisch Elisabeth , de Besi Patrizia , Righetti Pier Giorgio
Published: May 2005

Lateral aggregation in presence of a hydrophilic polymer (e.g. 10 kDa polyethylene glycol) in the gelling solution (Righetti et al., Electrophoresis 1992, 13, 587–595) is not inhibited by high ionic strength nor in the pH 4–10 interval. However, the bundles are disaggregated by glycerol (Tm at 20 %) and by ethylene glycol (Tm at 24.5 %) as well as by pH extremes (pH 3 and pH 11). Supercoiling is also strongly inhibited in a copolymer, formed by acrylamide an N,N‐dimethylacrylamide or N‐methylacrylamide. A level of 50 % uncoiling is obtained well before a 1:1 ratio, already at a level of 18 % N,N‐dimethylacrylamide. All the above data strongly suggest that the nascent chains are held together in bundles by hydrogen bonds prior to the cross‐linking event, instead of having a random orientation and distribution in the solvent. However, it is not possible to distinguish between H‐bonds oriented perpendicular to the chain axis vs. H‐bonds occurring within a single polymer filament, and the two types of H‐bonds probably coexist. Macroporous gels perform well in steady‐state electrophoretic techniques, such as conventional isoelectric focusing and immobilized pH gradients, where a large‐pore structure is necessary for fast protein migration and for attainment of equilibrium conditions.
Dissociation and electrophoretic separation of dextranase and dextranase inhibitor from a tighly bound enzymeinhibitor complex of Streptococcus sobrinus
Authors: Wellington Janet E. , Shaw Janet M. , Walker Gwen J.
Published: May 2005

Endodextranase was separated from dextranase inhibitor in culture filtrates of Streptococcus sobrinus by sodium dodecyl sulphate‐polylamide gel electrophoresis (SDS‐PAGE) in gel slabs containing blue dextran. Sample preparation included dissociation of the enzyme from its inhibitor by boiling for 1 min in SDS. During subsequent incubation of the gel, dextranase was located as clear bands on a blue background, and dextranase inhibitor appeared as blue zones on a clear background following incubation in dextranase solution. The enzyme and the inhibitor existed in multiple forms, and the range of molecular masses for dextranase (223–132 kDa) permitted an excellent separation from dextranase inhibitor (49–25 kDa). Although dextranase‐negative mutants, and wild type strains grown at low dilution rate in the chemostat, were devoid of free dextranase activity, the enzyme was easily located by analytical SDS‐PAGE. Likewise, analysis of filtrates from wild type strains, which contained no free inhibitor activity when growth occurred at high dilution rate, revealed dextranase inhibitor activity on the gels. The total production (free + combined) of dextranase and inhibitor by S. sobrinus was determined by dissociation of enzyme‐inhibitor complexes in concentrated cell‐free filtrates, their separation by preparative SDS‐PAGE and electroelution from the gels, followed by renaturation of protein activity. From a comparison of activity tests of free dextranase and free inhibitor in untreated filtrates with the results of similar tests on renatured electroeluates, the proportion of each constituent bound into a complex under each growth condition could be deduced.
Classification of mouse liver proteins by immobilized metal affinity chromatography and two‐dimensional electrophoresis
Authors: Jungblut Peter , Baumeister Hans , Klose Joachim
Published: May 2005

Mouse liver proteins were classified into metal‐binding and non‐binding proteins by combining immobilized metal ion affinity chromatography (IMAC) and twodimensional electrophoresis (2‐DE). The proteins were fractionated by three metal ions, Zn2+, Ni2+ and Cu2+, immobilized on iminodiacetic acid and then separated by 2‐DE. The total number of protein spots resolved by 2‐DE increased approximately twofold when the proteins were prefractionated by IMAC. By establishing 2‐DE standard patterns, 371 proteins were selected and then characterized according to their specificity in binding the three different metal ions. Only 48 proteins did not bind to any of the three metal ions investigated. Cu2+ was the most efficient ion in binding different proteins (310) compared to the other metals. Cu2+ bound to 42 proteins specifically and to 268 proteins unspecifically. Both Zn2+ and Ni2+ showed specific affinity only to four proteins.
Quantitative applications and validation of the resolution of enantiomers by capillary electrophoresis
Authors: Altria Kevin D. , Goodall David M. , Rogan Manus M.
Published: May 2005

The separation and quantitation of enantiomers is an important application of capillary electrophoresis. This paper reviews the performance and validation of capillary electrophoretic methods for this application. Limits of detection of < 0.1% are shown for determinations of minor enantiomer levels. Application areas include enantiomeric purity testing of pharmaceuticals and herbicides, reaction rate monitoring, stability testing, and clinical analysis. It is concluded that the number of quantitative application areas will undoubtedly expand as capillary electrophoresis becomes more widely established as an alternative and complement to high‐performance liquid chromatography.
A computer simulation accounting for dissimilar electrophoretic behavior between two similarly curved DNA fragments due to a difference in arc‐length
Authors: Wheeler David L. , Chrambach Andreas
Published: May 2005

Arc‐shaped bent DNA fragments of the same predicted planar curvature but differing in length by 20% were compared in regard to their mobilities in 3 to 10% polyacrylamide. The longer (155 bp) fragment is retarded far more severely than the shorter (124 bp) fragment. The effect of gel concentration in promoting the retardation is far more pronounced for the 155 bp than for the 124 bp fragment. Moreover, a temperature change from 25°C to 4°C does not substantially affect the gel concentration dependent mobility of the 124 bp fragment while it increases the retardation of the 155 bp fragment greatly. The strong increase in retardation brought about by a mere 20% increase in the length of the arc was accounted for by a simple computer simulation of gel electrophoresis which considered the rate of passage of arc‐shaped objects through a two‐dimensional array of disc‐shaped obstacles. Since the simulation relies exclusively on geometric factors, its success in predicting the behavior of the 124 and 155 bp DNA fragments suggests that geometric factors are largely responsible for their electrophoretic properties. The simulation can account for the strong temperature effect on the retardation of a model of the 155 bp DNA in polyacrylamide gels by showing that a decreased degree of random motion has a profound effect on the modeled 155 bp particle, but not on the modeled 124 bp DNA.
On the use of dimensionless parameters in acid‐base theory VII. The pH of solutions of salts of a strong acid and a weak base or vice versa
Authors: Rilbe Harry
Published: May 2005

The equation for the hydrogen ion activity in solutions of salts of a strong acid and a weak base, or vice versa, is of the thírd degree. Nevertheless, explicit equations for pH of such solutions can be formulated as linear functions of the logarithm of the salt concentration. The equations are valid within the major parts of pH and concentration intervals of experimental interest. The ranges of validity are given in terms of mathematically defined pH intervals. Close relations with pH of free base or free acid solutions of the same concentration as the salt are demonstrated. For instance, the pH difference between solutions of the salt and the free base or acid having unit activities is ± 7 pH units.
Immobilised ph gradient isoelectric focusing of wool proteins
Authors: Herbert Benjamin R. , Woods Joy L.
Published: May 2005

Conventional carrier ampholyte‐isoelectric focusing is unsuitable for separating wool and hair proteins. The inability to form stable narrow pH gradients at acidic pH values does not allow good resolution of the many acidic wool and hair proteins. To evaluate the usefulness of immobilised pH gradient‐isoelectric focusing (IPG‐IEF) for wool proteins, S‐amidomethyl wool proteins were analysed using IPG‐IEF. Over twenty bands were visible on a pH 4–7 IPG‐IEF gel. IPG‐IEF was combined with sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) to give a two dimensional separation, and over 50 protein spots were observed. IPG‐IEF appears to be ideally suited to the separation of wool proteins. The resolution of two‐dimensional electrophoresis using IPG‐IEF was greater than previously obtained using acid or alkaline PAGE in the first dimension. Good reproducibility of spot position was observed.
Comparison of protein analysis between embryonic and extraembryonic tissues during the 11th day of gestation of the mouse
Authors: Baumgartner Bernhard G. , Murach Karl‐friedrich , Schlegel Elisabeth , Praxmayer Claudia , Illmensee Karl
Published: May 2005

At day 11 of gestation, embryos and their extraembryonic tissues were isolated from the uterus of Him OF1/SPF mice and incubated in Dulbecco's modified Eagle's medium (DMEM) containing L‐[35S]methionine. After 4 h of incubation, the embryos were dissected to obtain the heart, liver, limb buds, and brain. The latter was fragmented into the telencephalon, mesencephalon, and myelencephalon. These organs and the extraembryonic tissues such as chorion, yolk sac, and placenta were processed for two‐dimensional (2‐D) gel electrophoresis. About 1000 proteins with relative molecular weights (Mr) varying from 10 000 to 200 000 and isoelectric points ranging from 4 to 10 could be detected on these gels. The protein patterns of the various organs and tissues were analyzed for organ‐ and cell lineage‐specific protein spots. We detected subtle differences in the protein patterns of the three cerebral areas when compared to each other. In addition, we found protein spots characteristic for the entire brain. We also found several heart‐specific protein spots. Distinct protein synthesis was also detected in liver and limb buds. Several groups of protein spots seem to be differentially regulated in these organs. Substantial differences between the patterns of embryonic and extraembryonic tissues were observed. In addition, several clusters of protein spots of well‐defined molecular weight could be detected only in extraembryonic tissues. We propose that organ‐ and tissue‐specific differences in protein synthesis are linked to some of the morphogenetic and functional processes during mammalian embryogenesis. Identification of particular proteins will serve as a basis to search for the corresponding genes.
Application of micellar electrokinetic capillary chromatography for monitoring of hippuric and methylhippuric acid in human urine
Authors: Lee Kong‐Joo , Lee Jong Jin , Moon Dong Cheul
Published: May 2005

The factors affecting micellar electrokinetic capillary chromatographic separation of hippuric and o‐, m‐, p‐methylhippuric acid were investigated by changing the species of micelles, and adding urea to the micellar solution. The analysis of hippurates in human urine is demonstrated under optimum conditions using 20 mM phosphate buffer (pH 8.0) containing 100 mM dodecyltrimethylammonium bromide and 4 M urea at −22 kV applied voltage. This method proved suitable for the screening of hippurates in human urine following occupational exposure to toluene and xylene.
Electrophoretic analysis of coniferyl alcohol oxidase and related laccases
Authors: Udagama‐Randeniya Preethi , Savidge Rodney
Published: May 2005

Gradient gel electrophoretic methods enabled a distinction to be made between coniferyl alcohol oxidase (CAO) of lignifying cell walls and a pI ∼ 9 pine “laccase” recently implicated in lignification (Science 1993 260, 672). Following treatment of a partially purified protein mixture from developing xylem of Pinus strobus with 2‐[N‐morpholine]ethanesulfonic acid (MES) buffer, isoelectric focusing and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis indicated that CAO had been selectively precipitated by MES and thereby purified to electrophoretic homogeneity. Purified CAO was determined to be a cell‐wall‐bound glycoprotein (38% glycan), Mr 107 500, pI,7.6, pH and temperature optima 6.3 and 30°C, respectively. By graphite‐furnace atomic‐absorption analysis, CAO contained one copper atom per protein molecule. Proteins obtained from lignifying cambial derivatives of conifers (family Pinaceae) and from Rhus typhina bark were compared with CAO and the pI ∼ 9 pine “laccase” following electrophoresis and Western blotting. For Abies balsamea, Larix laricina, Picea rubens, Pinus banksiana, Pinus taeda, and R. typhina, the isoelectric points of oxidatively active bands were identical to those of purified CAO. In addition, for all species only the pI 7.6 band was immunoreactive with antibodies against periodate‐deglycosylated CAO.
Capillary zone electrophoresis of oligosaccharides derivatized with various aminonaphthalene sulfonic acids
Authors: Chiesa Claudia , O'Neill Roger A.
Published: May 2005

Malto‐oligosaccharides were derivatized via their reducing ends with different aminonaphthalene mono‐, di‐ and trisulfonic acids by reductive amination. The derivatives were then separated by capillary zone eléctrophoresis in uncoated fused silica capillaries, using 50 mM triethylammonium phosphate buffer, pH 2.5, as running electrolyte. The effect of degree of charge on speed of analysis and resolution was studied for different aminonaphthalene mono‐, di‐ and trisulfonic acids. Under the conditions used, a higher degree of charge on the derivatives provided both faster analyses and higher resolution. Investigation of the electrophoretic behavior of derivatized oligosaccharides obtained from bovine pancreatic ribonuclease B gave insight into the possibility of applying such electrophoretic systems to the analysis of more complex carbohydrates. The resolution of positional isomers under the conditions described indicated that the high resolving power of this technique allows separations not strictly based on the effects of charge and mass of the analytes, but on structural characteristics as well. The relationship between electrophoretic mobility and molecular structure was investigated for the different derivatives.
Positional reproducibility of protein spots in two‐dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focusing in the first dimension: An interlaboratory comparison
Authors: Corbett Joseph M. , Dunn Michael J. , Posch Anton , Görg Angelika
Published: May 2005

An intra‐ and interlaboratory comparison of positional reproducibility of protein spots in two‐dimensional electrophoresis using immobilised pH gradients (IPG) in the first dimension (IPG‐DALT) was made. Aliquots of two different samples, human cardiac and barley leaf proteins, were separated in two different laboratories (London and Munich), using 180 mm long IPG gel strips, pH 4–8, for the first dimension and homogeneous SDS‐PAGE gels (12% T) for the second dimension. Subsets of 340 (cardiac) and 200 (barley) well‐resolved spots distributed across the 2‐D gel patterns were selected for computer analysis (PDQUEST) of positional reproducibility. The IPG‐dimension was highly reproducible in each laboratory, with a mean standard deviation of about 1 mm for both types of sample. Interlaboratory comparisons revealed identical results for barley with a mean standard deviation along thex‐axis of about 1 mm, whereas the cardiac matchset showed slightly more variability (mean standard deviation ∼ 1.5 mm). Nevertheless, IPG‐DALT provides significantly improved reproducibility of spot positions compared to conventional isoelectric focusing with synthetic carrier ampholytes.
Gel electrophoresis of end‐labeled DNA II. Dynamics and detrapping in pulsed fields
Authors: Défontaines Anne‐Dominique , Viovy Jean‐Louis
Published: May 2005

A theory for field‐inversion gel electrophoresis of a flexible polyelectrolyte bearing an uncharged bulky label at one end is described, and the evolution of the mobility with chain length, field strength, friction of the label, and the duration of the forward and reverse pulses is predicted. A new critical size, N detrap, is introduced, and its value calculated. It increases roughly linearly with the duration of the reverse pulses. Chains smaller than Ndetrap are detrapped by reverse pulses, and may have a high mobility, whereas chains larger than N detrap are not trapped, and have a very small mobility. This leads to an increase of the mobility (as compared with constant field) in a given range of sizes, and to a strong selectivity around N detrap Depending on the parameters, numerous other effects, including a secondary mobility plateau and band inversion, may appear. The corresponding regimes are predicted and discussed. All predictions are qualitatively consistent with available experimental data. We use them to suggest efficient conditions for the development of pulsed‐field trapping electrophoresis, a possible tool for improved DNA sequencing. In particular, we recommend using a ramping of pulse times, with a constant ratio of forward to reverse time in the range 3 to 5.