bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

AbstractTo 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

AbstractA 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

AbstractAn 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

AbstractExact 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

AbstractProteins 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

AbstractHigh 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

AbstractThe 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

AbstractIsoelectric 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

AbstractThe 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

AbstractWe 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

AbstractA 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

AbstractTo 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

AbstractThe 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

AbstractDetails 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

AbstractCapillary 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

AbstractIsoelectric 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

AbstractThis 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.