Discontinuous Buffer Research Articles

The Tip of the Coiled-coil Rod Determines the Filament Formation of Smooth Muscle and Nonmuscle Myosin

Myosin II self-assembles to form thick filaments that are attributed to its long coiled-coil tail domain. The present study has determined a region critical for filament formation of vertebrate smooth muscle and nonmuscle myosin II. A monoclonal antibody recognizing the 28 residues from the C-terminal end of the coiled-coil domain of smooth muscle myosin II completely inhibited filament formation, whereas other antibodies recognizing other parts of the coiled-coil did not. To determine the importance of this region in the filament assembly in vivo, green fluorescent protein (GFP)-tagged smooth muscle myosin was expressed in COS-7 cells, and the filamentous localization of the GFP signal was monitored by fluorescence microscopy. Wild type GFP-tagged smooth muscle myosin colocalized with F-actin during interphase and was also recruited into the contractile ring during cytokinesis. Myosin with the nonhelical tail piece deleted showed similar behavior, whereas deletion of the 28 residues at the C-terminal end of the coiled-coil domain abolished this localization. Deletion of the corresponding region of GFP-tagged nonmuscle myosin IIA also abolished this localization. We conclude that the C-terminal end of the coiled-coil domain, but not the nonhelical tail piece, of myosin II is critical for myosin filament formation both in vitro and in vivo.

Construction of a genetic map of barley (Hordeum vulgareL.) cross 'Azumamugi' × 'Kanto Nakate Gold' using a simple and efficient amplified fragment-length polymorphism system

We have devised a simple and efficient amplified fragment-length polymorphism (AFLP) system consisting of small slab gels, a discontinuous buffer system, and silver staining. Using this system, a single worker developed a barley map with 227 polymorphic fragments in 2 months. As a mapping population, 99 recombinant inbred lines of barley cultivars 'Azumamugi' x 'Kanto Nakate Gold' were used. Most of the 227 AFLP fragments showed a Mendelian segregation ratio of 1:1, and all were assigned to the seven barley chromosomes. Thus, these fragments are useful as molecular markers. They were integrated with 40 previously characterized sequence-tagged sites, 3 isozymes, and 2 morphological markers to construct an integrated map. The resulting map covered 925.6 cM with 272 markers (detecting 150 loci) at an average interval of 6.5 cM/locus. This system greatly simplifies map construction.

Protein aggregation mediated by cysteine oxidation during the stacking phase of discontinuous buffer SDS-PAGE.

The resolution of complex protein mixtures by discontinuous buffer SDS-PAGE is accomplished by their concentration into thin bands in the stacking gel, followed by their separation during migration through the resolving gel. Recombinant human interferon-inducible protein-10 (IP-10), a 10-kDa C-X-C chemokine with four cysteines, aggregated during the stacking phase of SDS-PAGE and generated a band with an apparent molecular mass of 18 kDa. This aggregation depended on the presence of reduced sulfhydryl residues on IP-10, on the amount of loaded protein, and on the concentration of the ammonium persulfate used to polymerize the stacking gel. The aggregation of IP-10 could be prevented by reduction of its sulfhydryls with dithiothreitol followed by irreversible blockade with iodoacetamide. These methods may be useful in the prevention of aggregation of sulfhydryl-containing proteins during SDS-PAGE, especially when large quantities are analyzed to assess their purity.

Allozyme and DNA sequence comparisons of nine species of Encephalartos (Zamiaceae).

Phylogenetic relationships between Encephalartos altensteinii Lehmann, E. arenarius R.A. Dyer, E. horridus (Jacquin) Lehmann, E. latifrons Lehmann, E. lehmannii Lehmann, E. longifolius (Jacquin) Lehmann, E. princeps R.A. Dyer and E. trispinosus (Hooker) R.A. Dyer were studied, using E. ferox Bertoloni f. as outgroup. Three continuous and one discontinuous buffer systems were used and gene products of 14 enzyme coding loci were examined by horizontal starch gel-electrophoresis. Genetic variation was studied in a cultivated population of E. lehmannii and the average heterozygosity value for this population is 13.5%, which falls within the range reported for other cycad species. Fixed allele differences between the species studied was not found at any of the loci studied, which suggest that these species are closely related. DNA sequence analysis of rbcL and ITS 1 & 2 genes (1428 and 895 basepairs, respectively) confirmed the close genetic relationships between these taxa. According to ITS and rbcL sequences E. altensteinii and E. princeps are sibling taxa which form a sister group to E. arenarius, E. horridus, E. latifrons, E. lehmannii, E. longifolius, and E. trispinosus. The genetic distances between both groups were 0.12-0.47% for ITS and 0.08-0.16% for rbcL DNA. The results indicate recent (probably pleistocenic) speciation for this group of cycads, and the relationships are discussed with reference to affinities based on morphology and distribution.

Electrophoretic separations in PMMA capillaries with uniform and discontinuous buffers

AbstractThe basic phenomena occurring within polymethylmethacrylate (PMMA, Plexiglass) capillaries under the conditions of capillary zone electrophoresis (CZE), isotachophoresis (CITP), and isoelectric focusing (CIEF) have been explored in our laboratory and are compared to those characteristic for untreated fused‐silica (FS) capillaries. The origin of charge at the inner wall of the PMMA capillary is different compared to that of FS, this having a significant impact on the magnitude of the electroosmotic flow. While both column materials are characterized with a negative surface charge, electroosmosis in PMMA is considerably smaller over the entire pH range. Furthermore, electroosmosis in PMMA and FS is shown to follow different dependencies on the ionic strength (I). Using a set of experimentally determined values, the electroosmotic mobility (μEO) of a PMMA capillary is best described using the relationship μEO=a+b/√I. For a FS capillary, μEO follows the expression μEO=a+b log(I). A dynamic capillary electrophoresis simulation model comprising the determined pH and ionic strength dependent wall titration data as input for calculation of electroosmosis is shown to provide CZE and CITP electropherograms that qualitatively agree well with those obtained experimentally. In contrast to FS and glass, electroosmosis at any pH in PMMA is too weak to perform bidirectional (i.e., simultaneous cationic and anionic) CZE and CITP analyses with a detector placed toward the cathodic capillary end. Furthermore, the same is true for the performance of CIEF with electroosmotic zone displacement. Imposed flow is demonstrated to provide the required net buffer flow. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 69–83, 2001

On the utility of an instrumented impact set-up to characterise the response of discontinuous buffer strips in carbon–epoxy systems in dry and wet conditions

The use of an instrumented impact test set-up to evaluate the influence of water ingress on the impact response of a carbon–epoxy (C–E) laminated composite system containing discontinuous buffer strips (BS) has been examined. The data on the BS-free C–E sample in dry conditions are used as reference to compare with the data derived from those immersed in water. The work demonstrated the utility of an instrumented impact test set-up in characterising the response, first owing to the architectural difference due to introduction of buffer strips and then due to the presence of an additional phase in the form of water ingressed into the sample. The presence of water was found to enhance the energy absorption characteristics of the C–E system with BS insertions. It was also noticed that with an increasing number of BS layer insertions, the load–time plots displayed characteristic changes. The ductility indices (DI) were found to display a lower value for the water immersed samples compared to the dry ones.

Stacking and discontinuous buffers in capillary zone electrophoresis

Discontinuous buffers for capillary zone electrophoresis (CZE) can be used under less rigid conditions compared to those for isotachophoresis for stacking. They can be prepared simply by modifying the sample itself, either by addition of small inorganic ions, low conductivity diluents, or both, and also by adjusting its pH, meanwhile injecting a large volume on the capillary. Zwitterionic and organic-based buffers such as triethanolamine and tris(hydroxymethyl)aminomethane (Tris) are well suited for stacking due to their low conductivity, provided the buffer is discontinuous as demonstrated here. A simple mechanism based on discontinuous buffers is described to explain many of the observed stacking types in CZE, pointing out the many similarities to transient isotachophoresis.

Stacking Neutral Analytes in Capillary Electrokinetic Chromatography with High-Salt Sample Matrixes

We recently described a method to stack neutral analytes in electrokinetic chromatography dependent upon using high-salt sample matrixes (Palmer et al. Anal. Chem. 1999, 71, 1679−1687) The use of a high-mobility co-ion in the sample matrix at a higher concentration than the like-charged electrokinetic vector (e.g., charged micelle) in the separation buffer as we previously suggested induces a single-boundary, discontinuous buffer system which subsequently stacks neutral analytes. Under these conditions, it is possible to electrokinetically inject neutral analytes directly from a high-salt sample matrix via electroosmotic flow, with stacking initiated simultaneously with injection. The correspondence by Quirino et al. (Anal. Chem., previous paper in this issue) suggests the effects of the high-salt sample matrix on stacking of micelles is obviated by a destacking of the micelle zone as it exits the sample zone. They reevaluate the high-salt stacking mode in terms of the γ function. Based on the present data, it is our interpretation that the stacking efficiency is not accurately represented by the γ function and that neither micelle zone nor neutral analyte destacking occurs under conditions we previously described. Low stacking efficiency when low-conductivity sample matrixes are used indicates that the high-salt stacking effect cannot be explained simply as a function of the k value of an analyte/micelle system.

Electrophoretic focusing preconcentration technique using a continuous buffer system for capillary electrophoresis

Detection and analysis of dilute small volume samples can be achieved by preconcentration techniques. Published techniques use specialized discontinuous buffer systems (sample stacking, field-amplification, and isotachophoresis) or incorporation of a chromatographic preconcentration chamber–capillary. By carefully exploiting flow and electric fields, preconcentration of analytes can be achieved without the need for discontinuous buffer systems or physical chromatographic devices. This focusing is achieved by independently controlling pressure flow and electrophoretic migration of analytes. Initiation of a voltage field at the immediate entrance of the capillary combined with adjusting bulk flow equal and opposite to the electrophoretic migration of the analytes results in preconcentration. Data are presented indicating an increase in local concentration of 200 nm carboxylate modified latex spheres within the immediate volume of the capillary entrance (specifically ∼15 pL) using laser-induced fluorescence detection. ©2000 John Wiley & Sons, Inc. J Micro Sep 12: 98–106, 2000

Miniaturized ultrathin slab gel electrophoresis with thermal lens microscope detection and its application to fast genetic diagnosis.

A miniaturized ultrathin slab gel electrophoresis (MUSGE) apparatus was developed, and fast separation of DNA fragments was obtained using it. To obtain sufficient separation efficiency in a limited space, a discontinuous buffer system was used. In general, it is difficult to cast a discontinuous ultrathin slab gel of adequate quality. However, the miniaturized resolving gel could be cast by taking advantage of the "capillary phenomenon" of the ultrathin channel. A gradient plate was used to control the height of the resolving gel and to form a clear interface between the concentrating gel and the resolving gel. This method was used to cast multiple gels simultaneously and reproducibly. The gradient plate also facilitated sample introduction, which was carried out by using a micropipet. A 25-mm-long and 80-micron thick-resolving gel was used to separate the 100-base pair ladder DNA within 10 min. Bandwidth was reduced to 100-200 microns, thus improving the number of theoretical plates to 22,000, which was comparable to that in conventional slab gel electrophoresis even though the migration distance was reduced to 1/10. Satisfactory lane-to-lane reproducibility (RSD < 1.0%, n = 6) and gel-to-gel reproducibility (RSD < 2.7%, n = 4) were obtained. Finally, the MUSGE apparatus was successfully applied to get a rapid genetic diagnosis.

Fast Slab Gel Electrophoretic Separation of DNA Fragments with a Short Migration Distance Using Thermal Lens Microscope.

We demonstrated fast separation of DNA fragments in a short slab gel through the minimization of band-broadening. A discontinuous buffer system was used to reduce band-broadening from sample introduction, and a high spatial resolution thermal lens microscope (TLM) was used to reduce such an effect from the detector. Bands of DNA fragments were sharpened into 100 m m, which was about 1/10 of the bandwidths in conventional continuous slab gel electrophoresis. The 100 base pair ladder DNA sample, which ranges from 100 bp to 2000 bp, was completely separated within 15 min with a migration distance of 18 mm and was detected by TLM after silver staining. These results suggested the possibility of fast separation of DNA fragments in a miniaturized discontinuous slab gel. As well as the inherent properties of parallel separation of slab gel, high throughput separation can be obtained, which is significant for DNA analyses such as DNA sequencing, genetic diagnosis and forensic identification analyses.

Phylogenetic relationships, based on allozyme data, between six cycad taxa indigenous to South Africa

Phylogenetic relationships between Encephaiartos altensteinii Lehmann, E. friderici-guilielmii Lehmann, E. lehmannii Lehmann, E. natalensis Dyer & Verdoorn, E. transvenosus Stapf & Burtt Davy and E. villosus Lemaire were studied, using Cycas revoluta Thunberg as outgroup. Three continuous and one discontinuous buffer systems were used and gene products of 21 enzyme coding loci were examined by horizontal starch gel-electrophoresis. A biochemical key, based on fixed allele differences, is presented. Fixed allele differences at one locus between E. altensteinii and E. natalensis may confirm that these species do not share the same gene pool. However, the genetic distance is the least (0.042) between these two species, compared to the mean genetic distance value of 0.222 for the other ingroup taxa. The results are discussed with reference to affinities based on morphology and distribution.

Modeling of the Impact of Ionic Strength on the Electroosmotic Flow in Capillary Electrophoresis with Uniform and Discontinuous Buffer Systems

A new dynamic computer model permitting the combined simulation of the temporal behavior of electroosmosis and electrophoresis under constant voltage or current conditions and in a capillary which exhibits a pH-dependent surface charge has been constructed and applied to the description of capillary zone electrophoresis, isotachophoresis, and isoelectric focusing with electroosmotic zone displacement. Electroosmosis is calculated via use of a normalized wall titration curve (mobility vs pH). Two approaches employed for normalization of the experimentally determined wall titration data are discussed, one that considers the electroosmotic mobility to be inversely proportional to the square root of the ionic strength (method based on the Gouy-Chapman theory with the counterion layer thickness being equal to the Debye-Hückel length) and one that assumes the double-layer thickness to be the sum of a compact layer of fixed charges and the Debye-Hückel thickness and the existence of a wall adsorption equilibrium of the buffer cation other than the proton (method described by Salomon, K.; et al. J. Chromatogr. 1991, 559, 69). The first approach is shown to overestimate the magnitude of electroosmosis, whereas, with the more complex dependence between the electroosmotic mobility and ionic strength, qualitative agreement between experimental and simulation data is obtained. Using one set of electroosmosis input data, the new model is shown to provide detailed insight into the dynamics of electroosmosis in typical discontinuous buffer systems employed in capillary zone electrophoresis (in which the sample matrix provides the discontinuity), in capillary isotachophoresis, and in capillary isoelectric focusing.

Variação Polimórfica de Hemoglobinas em Búfalos (Bubalus bubalis)

No presente trabalho foi estudado o polimorfismo bioquímico das hemoglobinas de 96 búfalos indianos de diferentes cruzamentos envolvendo as raças Murrah, Jafarabadi e Mediterrâneo, criados na Fazenda Experimental Lageado - UNESP - Botucatu, Estado de São Paulo. As variantes de hemoglobinas foram identificadas através de eletroforese em gel de Agar-Amido em sistema de tampão descontínuo, pH 8,6. Animais portando uma banda rápida (A,) foram considerados AA (4,4%); búfalos com duas bandas (A, e A2) foram considerados AB (31,87%) quando A2 era mais fraca e BB (31,87%) quando A2 era mais forte. Uma terceira banda foi encontrada, chamada N. Fenótipos com três bandas foram encontrados com as respectivas freqüências: ABN (3,3%) e BBN (30,77%). Análises densitométricas e a falta de animais AAN levaram à conclusão que, de alguma forma, a síntese da cadeia p mutante que origina a banda N esteja relacionada provavelmente ao alelo a". Provavelmente, a alta freqüência de animais com banda N seja devida ao uso intenso de poucos reprodutores.

Mnium orientale sp. nov. from Japan Is Morphologically and Genetically Distinct from M. hornum in Europe and North America

Mnium orientale sp. nov. from Japan Is Morphologically and Genetically Distinct from M. hornum in Europe and North America

Capillary gel electrophoretic separation of DNA restriction fragments in a discontinuous buffer system

Separation of single and double stranded DNA molecules by capillary gel electrophoresis has a rapidly growing interest. Similar to the polycrylamide slab gel based separation methods, in capillary gel electrophoresis, the paek/bans spacing usually decreases with the increasing size/length of the DNA molecule. Additionally, employing the regularly used Tris-borate buffer system, fronting peaks are often obtained. By the application of an electrolyte step gradient during capillary gel electrophoretic separation of dsDNA molecules, the apparent peak shape can be improved and the required analysis time decreased.

Electrophoresis of Polyethylene Glycols and Related Materials as Sodium Dodecyl Sulfate Complexes

Polyethylene glycol (PEG) and polyethylene glycol derivatives are analyzed by a modification of the sodium dodecyl sulfate–polyacrylamide stacking gel electrophoresis procedure of Kurfürst. Gels using a discontinuous buffer system but which do not have a separate stacking gel are used without loss of resolution and with less tendency to form artifactual multiple or distorted bands. Examination of several commercial preparations of PEGs and PEG derivatives on such gels indicates heterogeneity other than the expected unimodal size distributions. SDS–gel electrophoresis of proteins or other materials in samples containing PEGs may yield gels with zones of contamination from the PEGs. Methods of reducing such contamination are suggested.

Reproducibility of mobility in gel electrophoresis.

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra-experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra- and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra-experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS-proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the "nonrestrictive" stacking gel.

General Protein (P-3) Polymorphism in Honey Bee (Apis mellifera L.) from Central Anatolia

The General Protein (P-3) system was investigated in 15 honey bee (A. mellifera L.) colonies from 3 different localities in this study. Horizontal starch gel electrophoresis was performed on dark-eyed pupae (13-19 days old) using Poulik's (1) discontinuous buffer system. The frequencies of P-3F allele in three apiaries were 0.658, 0.333 and 0.442.

Identification, Isolation, and Cloning of a Bacillus thuringiensis CryIAc Toxin-binding Protein from the Midgut of the Lepidopteran Insect Heliothis virescens

Bacillus thuringiensis toxins are insecticidal to a variety of insect species. The selectivity of the toxins produced by these bacteria is dependent on both the toxin structure and the receptor sites that are present in different insect species. One of these toxins, CryIAc, is highly insecticidal to the noctuid pest Heliothis virescens. Using toxin overlay assay, a 120-kDa glycoprotein was identified as a toxin-binding protein. This protein was partially purified, its N-terminal sequence was determined, and the full-length cDNA encoding this protein was isolated from a H. virescens midgut library. The B. thuringiensis toxin-binding protein, BTBP1, has high homology to aminopeptidase N from eukaryotes and prokaryotes.