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PubMedDate not supplied

[The sequential use of local vacuum magnetotherapy and papaverine electrophoresis with sinusoidal modulated currents in impotence].

105 patients with chronic nonspecific prostatitis were examined and treated with papaverin electrophoresis using sinusoidal modulated currents (SMC) and local vacuum magnetotherapy (LVMT). Papaverin SMC electrophoresis and LVMT stimulated cavernous circulation. The highest stimulation was achieved at successive use of LVMT and the electrophoresis. LVMT followed by the electrophoresis maintained good cavernous circulation for 5-6 hours after the procedure in the course of which several spontaneous erections were observed.

Adult↗
PubMed1981

Micromethod for detection of oligoclonal IgG in unconcentrated CSF by polyacrylamide gel electrophoresis.

A micromethod to detect oligoclonal IgG from 50 microL of unconcentrated CSF was developed by using polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE). Of 17 patients with multiple sclerosis, oligoclonal bands were demonstrated in 16 instances (94%) by micro-SDS-PAGE and in 13 (76%) by agarose gel electrophoresis. The corresponding figures among 30 patients with optic neuritis were 16 (54%) and five (17%), respectively, and among ten patients with other neurological disease the figures were two (20%) and none, respectively. Thus, micro-SDS-PAGE is more sensitive than agarose gel electrophoresis for detection of oligoclonal IgG. The small volume of unconcentrated CSF that is required enhances the usefulness of this test.

Adolescent↗
PubMed1999

Analysis of intact proteins from cerebrospinal fluid by matrix-assisted laser desorption/ionization mass spectrometry after two-dimensional liquid-phase electrophoresis.

A novel combination of methods, two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), have been used for the analysis of intact brain-specific proteins in cerebrospinal fluid (CSF). 2D-LPE is especially useful for isolating proteins present in low concentrations in complex biological samples. The proteins are separated in the first dimension by liquid-phase isoelectric focusing (IEF) in the Rotofor cell and in the second dimension by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in the Preparative cell. The removal of SDS by chloroform/methanol/water, followed by sample preparation with the addition of n-octylglucoside, easily interfaced 2D-LPE with MALDI-TOFMS for analysis of intact proteins. Further characterization by proteolytic digestion is also demonstrated. The knowledge of both the molecular weights of the protein and of the proteolytic fragments obtained by peptide mapping increases specificity for protein identification by searching in protein sequence databases. Two brain-specific proteins in human CSF, cystatin C and transthyretin, were isolated in sufficient quantity for determination of the mass of the whole proteins and their tryptic digest by MALDI-TOFMS. This approach simplified the interface between electrophoresis and MALDI-TOFMS.

Blotting, Western↗
PubMedDate not supplied

Identification of proteins in a human pleural exudate using two-dimensional preparative liquid-phase electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry.

Pleural effusion may occur in patients suffering from physical trauma or systemic disorders such as infection, inflammation, or cancer. In order to investigate proteins in a pleural exudate from a patient with severe pneumonia, we used a strategy that combined preparative two-dimensional liquid-phase electrophoresis (2-D LPE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Western blotting. Preparative 2-D LPE is based on the same principles as analytical 2-D gel electrophoresis, except that the proteins remain in liquid phase during the entire procedure. In the first dimension, liquid-phase isoelectric focusing allows for the enrichment of proteins in liquid fractions. In the Rotofor cell, large volumes (up to 55 mL) and protein amounts (up to 1-2 g) can be loaded. Several low abundance proteins, cystatin C, haptoglobin, transthyretin, beta2-microglobulin, and transferrin, were detected after liquid-phase isoelectric focusing, through Western blotting analysis, in a pleural exudate (by definition, >25 g/L total protein). Direct MALDI-TOF-MS analysis of proteins in a Rotofor fraction is demonstrated as well. MALDI-TOF-MS analysis of a tryptic digest of a continuous elution sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) fraction confirmed the presence of cystatin C. By applying 2-D LPE, MALDI-TOF-MS, and Western blotting to the analysis of this pleural exudate, we were able to confirm the identity of proteins of potential diagnostic value. Our findings serve to illustrate the usefulness of this combination of methods in the analysis of pathological fluids.

Blotting, Western↗
PubMed1999

Comparison between capillary and polyacrylamide gel electrophoresis for identification of Lolium species and cultivars.

In recent years variety discrimination has been achieved in a range of agricultural crops by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Previous works on genus Lolium have shown the effectiveness of this technique for the topic of cultivar identification. In the present research the potential of capillary gel electrophoresis (CGE) for identification of Italian (Lolium multiflorum Lam.), annual (Lolium rigidum Gaud.) and perennial (Lolium perenne L.) ryegrass cultivars in comparison with SDS-PAGE was investigated. Separation conditions of SDS-CGE were chosen in order to obtain electrophoretic data comparable with those of SDS-PAGE, at the expense of analysis speed. Both peak area and migration time of SDS-CGE electropherograms were reproducible. In the examined cultivars, a total of 27 (16.8-96.8 kDa) and 28 (13.2-111.5 kDa) protein subunits were detected by SDS-PAGE and SDS-CGE, respectively. The variability in seed storage protein composition was processed by numerical taxonomy. All cultivars were clearly identified by both electrophoresis systems. The orthogonality between SDS-PAGE and SDS-CGE was suggested by the lack of correlation among protein profiles obtained with the two separation systems. The nonredundant information from these analytical systems should provide a relevant benefit for identification of Lolium cultivars and wild biotypes that are extremely uniform phenotypically.

Electrophoresis, Capillary↗
PubMed2001

Field-inversion electrophoresis on a microchip device.

We have proposed a field-inversion electrophoresis on a microchip for a shorter effective length, and investigated the external frequency for the DNA analysis based on the field-inversion electrophoresis device. By using the optimized frequency, we demonstrated that the field-inversion electrophoresis has great potentials for the separation of DNA fragments with shorter effective length.

DNA, Single-Stranded↗
PubMed2001

Protein changes in post mortem sea bass (Dicentrarchus labrax) muscle monitored by one- and two-dimensional gel electrophoresis.

This study was devoted to the identification of specific peptides and proteins which can be used as indicators of freshness in fish. The post mortem evolution of protein patterns in farmed sea bass muscle was monitored by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) after 0, 2, 4, and 6 days cold storage. SDS-electrophoresis, of total proteins and proteins soluble in low-ionic-strength solutions, revealed the gradual disappearance of a protein band of 16 kDa immediately after fish death. 2-DE allowed the classification of fish samples according to post mortem time. Three spots of interest, which disappeared progressively, were identified on the 2-DE patterns. Further research is required to establish the identity of these polypeptides and to evaluate their expression and post mortem evolution in another fish species.

Animals↗
PubMed2002

The use of gel and capillary electrophoresis to investigate some of the fundamental physical properties of DNA.

Electrophoresis is a powerful technique which can be used not only for the size-based separation of DNA in slab gels and sieving liquid polymers, but also for the analysis of sequence-dependent variations in DNA conformation and structure. Polyacrylamide gels are useful for conformational analysis, because bent or curved DNA molecules migrate anomalously slowly in this gel medium. Bending is often (but not always) associated with runs of adenine residues (A-tracts) that occur in phase with the helix repeat. The unique structure responsible for DNA bending "melts out" at a temperature considerably below that of strand separation. The circular permutation assay is another polyacrylamide gel-based method of detecting bending. It has usually been applied to small restriction fragments, but can also be used to detect bending in plasmid-sized DNA molecules. The apparent bends in plasmid DNAs tend to be located near biologically important regions of the sequence, such as the origin of replication, the start site of transcription, and the promoters of certain genes. Finally, capillary electrophoresis in free solution (without sieving liquid polymers) can be used to determine the diffusion coefficients of small DNA molecules, detect DNA-buffer interactions, and analyze the sequence dependence of counterion binding. Counterions appear to be preferentially bound to DNA oligomers containing A-tracts, especially the A(n)T(n) sequence motif. Typical examples of these applications of gel and capillary electrophoresis to the study of DNA conformation and structure are described.

Acrylic Resins↗
PubMed2002

A hybrid microdevice for electrophoresis and electrochromatography using UV detection.

We have designed a new class of microdevices composed of a supporting plastic (polyvinyl chloride, PVC) plate integrated with a groove for a piece of fused silica capillary (the separation channel), a slit for on-tube detection, an "islet" for the application of sample, electrode vessels and platinum electrodes. The design permits electrophoretic, electrochromatographic and chromatographic separations with on-tube UV detection. The efficient heat dissipation allows relatively high field strengths. This article is the first one dealing with microdevices where polymer solutions are replaced by homogeneous gels. A new type of gels synthesized from acrylamide and 2-hydroxy-3-allyloxy-propyl-beta-cyclodextrin (allyl-beta-CD) as a cross-linker was employed for electrophoresis and electrochromatography. 2-Acrylamido-2-methylpropanesulfonic acid was added to the monomer solution to create a high electroendosmotic flow in electrochromatographic runs. These gels have excellent electrochromatographic and electrophoretic properties for low-molecular-weight compounds and DNA, as shown previously, namely high resolution combined with high stability. The unique cross-linker can be used for specific interaction with the alkyl and phenyl groups. The tripeptide glutathione (gamma-L-glutamyl-L-cysteinyl-glycine) and its benzyl conjugates were selected as model compounds to study the resolving power of the gel because they are difficult to separate by free zone electrophoresis. The limit of detection (LOD) for S-benzyl-glutathione was determined (ca. 7 microM). Run-by-run reproducibility was high (the separation factor of glutathione in the gel was 0.3 with 2.5% coefficient of variation, CV). Neutral compounds (acetone, acetophenone, propiophenone and butyrophenone) were separated electrochromatographically in the gel. The influence of organic solvent (acetonitrile) on the electroendosmotic mobility was similar to that in reversed-phase separations, although the separation mechanism is different. ATP, ADP and AMP were separated in less than 10 s by free-zone electrophoresis.

Chromatography, Micellar Electrokinetic Capillary↗
PubMed2002

Comparison of one-dimensional and two-dimensional gel electrophoresis as a separation tool for proteomic analysis of rat liver microsomes: cytochromes P450 and other membrane proteins.

Gel electrophoresis in combination with peptide mass fingerprinting is the method of choice for proteomic profiling of various in vitro and in vivo biological systems. In the investigation reported here we analyzed the protein composition of hepatic microsomes from untreated and phenobarbital treated rats, using one-dimensional (1-DE) and two-dimensional (2-DE) gel electrophoresis, followed by tryptic peptide mapping. To better characterize capabilities of 2-DE 1-DE with regard to microsomal membrane proteins, "ghosts" of microsomal vesicles enriched in membrane proteins were obtained and analyzed. Both 1-DE and 2-DE showed that phenobarbital induces not only cytochromes P450 2B1and 2B2 but such stress related endoplasmic reticulum proteins as protein disulfide isomerase A(3) and A(6) and 78 kDa glucose regulated protein. The analytical performance of 1-DE with regard to endoplasmic reticulum membrane proteins is incomparably greater than that of 2-DE. Twenty-two out of a total of thirty-four known to date microsomal rat membrane proteins were identified by 1-DE in combination with matrix-assisted laser desorption/ionization-mass spectrometry of in-gel digests. At the same time using various types of 2-DE, we were able to identify only three rat microsomal membrane proteins. The data presented in this manuscript clearly demonstrate that 1-DE in combination with peptide mass fingerprinting can be successfully used for cataloging proteins of the endoplasmic reticulum, and that the proteomic analysis of the subcellular organelles containing a considerable number of highly hydrophobic membrane proteins should be performed by combined application of 1-D and 2-D electrophoresis.

Animals↗
PubMed2002

Gradiflow as a prefractionation tool for two-dimensional electrophoresis.

The Gradiflow trade mark, a preparative electrophoresis instrument capable of separating proteins on the basis of their size or charge, was used to separate whole cell lysates, prepared from bakers yeast (Saccharomyces cerevisiae) and Chinese snow pea seeds (Pisum sativum macrocarpon), into protein fractions of different pH regions. Both broad and narrow range (with a difference of approximately 1 pH unit) pH fractions were obtained. Analysis of the protein fractions by isoelectric focusing gels and two-dimensional (2-D) polyacrylamide gel electrophoresis indicated minimal overlap between the pH fractions. Further, when the prefractionated acidic samples were analyzed on pH 4-7 immobilized pH gradient 2-D gels, improved resolution of the proteins within the chosen pH region was achieved compared to the unfractionated samples. This study demonstrates that the Gradiflow could be used as a preparative electrophoresis tool for the isolation of proteins into distinct pH fractions.

Electrophoresis, Gel, Two-Dimensional↗
PubMedDate not supplied

Transverse alternating field electrophoresis and applications to mammalian genome mapping.

The transverse alternating field electrophoresis system is a pulsed field gel apparatus that has been used to separate DNA molecules that range in size from a few thousand to approximately 7 million base pairs. This apparatus uses a vertical gel and a simple electrode arrangement to produce electric fields that are uniform across all lanes of the gel. The velocity of identical molecules does not vary from lane to lane, and hence there is no distortion in the paths of the DNA. The performance of this system is illustrated here using the chromosomes from S. pombe and S. cerevisiae, and restriction enzyme digested mammalian DNA. The mobility of molecules up to 1100 kilobase pairs is linear with size and can be accomplished in overnight runs. Resolution of very large molecules requires electrophoresis for several days, but molecules from 200 to 7000 kilobase pairs can be separated on a single gel. This electrophoresis system has been used extensively in the construction of a physical map of human chromosome 21, and examples of this application are discussed.

Animals↗
PubMed1991

Two-dimensional electrophoresis in small gels for applications in veterinary medicine.

A versatile method for two-dimensional electrophoresis that can be performed easily, even in small routine laboratories, is described. The procedure combines a first-dimensional isoelectric focusing run in a PhastSystem with denaturing electrophoresis in a small vertical electrophoresis chamber. The described arrangement of two first-dimensional gel strips in the second dimension allows direct comparison of two related samples, eliminating most of the artifacts that usually lead to misinterpretations. The presented procedure can be conveniently adapted to the needs of each laboratory; at present, it is being extensively used to establish protein patterns of healthy individuals of different species, a prerequisite with regard to help and support diagnosis.

Animals↗
PubMed1993

Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix.

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.

Avidin↗
PubMedDate not supplied

Studies on 16 kDa selenium-containing proteins enriched by means of preparative electrophoresis.

By in vivo labeling with [75Se]selenite and separation of the proteins in the tissue homogenates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), several selenium-containing proteins or protein subunits were detected in rat tissues (liver, lung, spleen and prostate). Their distribution among the cell components was investigated after fractionation by means of differential centrifugation. The selenium-containing proteins in the 16 kDa range were found to be mainly membrane-bound. By two-dimensional electrophoresis they were resolved into three labeled spots, two with the same relative molecular mass and pI values of about 4.8 and 5.0 and the third with a slightly lower molecular mass and a pI of 4.8. For further investigation they were concentrated and separated from the other labeled compounds by SDS-PAGE using preparative flow-through electrophoresis.

Animals↗
PubMed1993

Characterization of E-PHA-reactive alpha-fetoprotein isoforms by two-dimensional lectin affinity electrophoresis.

Erythroagglutinating phytohemagglutinin (E-PHA)-dependent isoforms of human alpha-fetoprotein (AFP) from cord blood were analyzed for their carbohydrate structures by two-dimensional electrophoresis with E-PHA combined with extended agarose gel electrophoresis or with affinity electrophoresis with concanavalin A or Allomyrina dichtoma lectin. By means of neuraminidase and/or beta-galactosidase treatment, AFP-P2 was identified as alpha 2-->6 disialo-AFP, AFP-P3 as having biantennary structures with alpha 2-->6 monosialylated galactose of the Mannose (Man) alpha 1-->6 arm, AFP-P4 as having alpha 2-->6 monosialylated galactose of the Man alpha 1-->3 arm, and AFP-P5 as disialo-AFP with alpha 2-->3 sialylated galactose of the Man alpha 1-->6 antenna with the alpha 2-->6 sialylated galactose of the other antenna. Desialylated AFP with the terminal galactose of the Man alpha 1-->6 antenna with or without the galactose of the other arm also had a migration of AFP-P4, and other hydrolytic intermediates without the terminal galactose of the Man alpha 1-->6 arm with and without the galactose of the other antenna had mobilities of AFP-P3s and AFP-P3, respectively. Thus, the present system of two-dimensional lectin affinity electrophoreses would provide a model for the determination of the sugar chain structure of glycoproteins.

Carbohydrate Conformation↗
PubMedDate not supplied

Ultrasensitive plasmid mapping by high performance capillary electrophoresis.

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 linear polyacrylamide 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.

Electrophoresis, Agar Gel↗
PubMedDate not supplied

Capabilities and potentialities of transverse pore gradient gel electrophoresis.

Transverse pore gradient gel electrophoresis is important as a tool for obtaining nonlinear Ferguson plots [log(mobility) vs. gel concentration], e.g. in application to DNA in polyacrylamide gels or to agarose gels, with the purpose of evaluating molecular properties (size, conformation, malleability) and gel fiber properties (fiber radius and length per unit volume). To date, it is capable of (i) yielding gel patterns ("Ferguson curves") of migration distance vs. predicted % T-range of the pore gradient, assuming its linearity; (ii) yielding information regarding molecular conformation from the intersection of Ferguson curves of unknowns (e.g. bent DNA) with those of standards; (iii) acquisition of Ferguson curves by computer, using prototype instrumentation; (iv) mathematical manipulation of acquired Ferguson curves to yielding Ferguson plots, providing that mobility in free solution has been assessed by capillary zone electrophoresis. The potentialities of the method remain unfulfilled to date due to (i) the unavailability, with a single exception, of an accurate and precise way to produce pore gradients of known shape; (ii) unavailability of a routinely applicable analysis for % T; (iii) unavailability of optimized, user-friendly and foolproof instrumentation for computer acquisition of Ferguson curves, including the present inapplicability of a commercially available electrophoresis apparatus with intermittent optical detection to transverse pore gradient gels; and (iv) unresolved problems in the statistical evaluation of Ferguson curves.

DNA↗