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PubMed2002

Stacking for nonaqueous capillary electrophoresis.

Nonaqueous capillary electrophoresis (NACE) is a useful mode in CE for separation and quantification of hydrophobic compounds. However, because of the low conductivity of most of the organic solutions, stacking is not used often in this technique and the sample volume is very limited. As a result of the small sample volume, the detection limits are poor. Furthermore, NACE is affected greatly by the presence of salts in the sample. Here, we show that transient isotachophoresis (t-ITP) can be used easily in this type of electrophoresis to enhance the detection limits and also to reverse the deleterious effects of salts in the sample. Several factors, which affect the stacking in this type of electrophoresis, are described. For example, the presence of salts in the organic solvent, type of sample introduction, and the solvent for the terminating ion were all found to have profound effects on the degree of concentration. Furthermore, the separation time can be shortened by t-ITP.

Electrophoresis, Capillary↗

PubMed2002

Electrophoresis of randomly charged particles.

The technique of "rotational electrophoresis" has been developed recently to measure the charge nonuniformity on Brownian particles in a suspension. Measurements of the angular velocity in electrophoresis are interpreted with electrokinetic theory to give a standard deviation of zeta potential among N regions on a particle. A key question is how various molecular mechanisms of charging could lead to charge nonuniformity on a length scale of O (100 nm). Simple statistical physics is used to show that although Poisson charging with univalent charges cannot explain recent measurements showing significant charge nonuniformity, random charging with multivalent charges can better explain the data. In addition, the result shows that a small number of charged "visualization" molecules can be adsorbed to a particle without impacting the experimental technique of rotational electrophoresis.

Colloids↗

PubMed2002

Chiral analysis of biogenic DL-amino acids derivatized by urethane - protected alpha-amino acid N-carboxyanhydride using capillary zone electrophoresis and micellar electrokinetic chromatography.

A new analytical method for enantioselective separation of DL-amino acids derivatized by N-fluorenylmethoxycarbonyl-L-alanyl N-carboxyanhydride (FMOC-L-Ala-NCA) using capillary electrophoresis was developed. Separation parameters, such as composition and pH of the background electrolyte, and concentration of gamma-cyclodextrin (in capillary zone electrophoresis) and sodium dodecyl sulfate (in micellar electrokinetic chromatography) were optimized. The separation method was validated and it suits well for purity analysis. Detection limit of the method was 0.2% of the minor enantiomer in the major one. The level of racemization in coupling during solid-phase peptide synthesis was studied using capillary electrophoresis with gamma-cyclodextrin as a chiral selector. The anchorage of the first (C-terminal) amino acid derivative to the solid supports bearing the hydroxylic groups is the key step of the synthesis affecting the extent of its racemization. FMOC-L-phenylalanine was chosen as the suitable model amino acid derivative making it possible to study the degree of racemization of N-fluorenylmethoxycarbonyl-L-alanine-L-phenylalanine synthesized on different polymer resins, using the different condensation agents.

Amino Acids↗

PubMed2002

Investigation of the possibility of geometrical electrophoresis.

We investigate the possibility of geometrical electrophoresis, which is based on nanofabrication techniques. (GEE) utilizes geometrical effects during electrophoresis, which are generated by physical interactions between walls and a macromolecule confined in spaces smaller than the Flory radius. When a polymer is injected into a small space, confinement energy is usually required. However, the confinement energy form depends on the geometry of the space. In the case of electrophoresis, the electric field itself changes depending on the geometry. Using a nanofabricated quartz chip with a curved channel, we investigated electrophoretic behavior of high molecular weight DNA based on the curvature effect.

DNA↗

PubMed2002

On-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis: a two-dimensional separation system for proteomics.

A microdialysis junction is employed as the interface for on-line coupling of capillary isoelectric focusing with transient isotachophoresis-zone electrophoresis in a two-dimensional separation system. Capillary isoelectric focusing not only provides high-resolution separation of tryptic peptides based on their differences in isoelectric point, but also potentially allows the analysis of low-abundance proteins with a typical concentration factor of 50-100 times. Carrier ampholytes, employed for the creation of a pH gradient during focusing, are further utilized as the leading electrolyte in the second separation dimension, transient isotachophoresis-zone electrophoresis. Many peptides which have the same isoelectric point would most likely have different charge-to-mass ratios, and thus different electrophoretic mobilities in zone electrophoresis. Two-dimensional separation of proteolytic peptides is demonstrated using standard proteins, including cytochrome c, ribonuclease A, and carbonic anhydrase II. The maximum peak capacity is estimated to be around approximately 1600 and can be significantly increased by simply increasing the capillary column length and manipulating the range of pH gradient in isoelectric focusing. In addition to enhanced separation efficiency and resolution, this two-dimensional electrokinetic separation system permits sensitive and comprehensive analysis of peptide fragments, especially when integrated with electrospray ionization mass spectrometry for peptide/protein identification.

Amino Acid Sequence↗

PubMed2002

Vanadium speciation in serum by means of blue native gel electrophoresis.

Slab-gel electrophoresis has been applied to the speciation of vanadium in serum. The electrophoresis separation is an adaptation of the blue native polyacrylamide gel electrophoresis separation necessary to ensure the stability of the vanadium-protein complex; Coomassie blue was used to shift the charges of the proteins and to stabilize the vanadium complex. The detection of the vanadium species was made possible by the use of the (48)V radiotracer and the phosphor-screen technology. The method was first developed using transferrin, incubated with (48)V, as a model. After it was proved that the vanadium-transferrin complex was stable during separation, the method was validated by separating serum incubated with (48)V. The efficiency of the separation was assessed according to two parameters: resolution and conservation of the species. First, the resolution of the separation was as expected from a native separation. Second, the release of free vanadium from the transferrin complex, which was the main vanadium species expected, was negligible, which proves that the species remain intact during separation. In accordance with the literature, it was found that vanadium binds to transferrin in incubated serum at these low concentrations.

Autoradiography↗

PubMed2002

High performance two-dimensional gel electrophoresis using a wetting agent Tergitol NP7.

Two-dimensional electrophoresis is an efficient method for the analysis of a broad range of complex protein samples. Current two-dimensional gel techniques are not suited for analysis of the small amount of proteins from tissue samples in the presence of high concentration of salts. Here we describe an improved two-dimensional gel electrophoresis procedure based on the use of a nonionic wetting agent, Tergitol NP7, in rehydration solution combined with the application of a linear potential sweep during isoelectrofocusing. This experimental approach yields a dramatic increase in the resolution and focusing of proteins visualized on two-dimensional gels. This technique is less time-consuming and laborious than the current techniques and can be used for a variety of two-dimensional electrophoresis applications, including proteome analysis.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1990

Electrophoresis and movements of fluorescence pattern after photobleaching of large DNA fragments in agarose gels.

By combining electrophoresis with movements of fluorescence pattern after photobleaching (MOFPAP), which is abbreviated as EMOFPAP, we are able to measure electrophoretic mobilities of large DNA fragments in an agarose gel within a fairly short time scale (about 10 min or even down to 1 min). The new method represents a significant improvement in experiment time when compared with the time (typically on the order of hours) required to determine the average electrophoretic mobility of large DNA fragments in agarose gels by means of either conventional gel electrophoresis or pulsed-field gel electrophoresis. In this article, we present the EMOFPAP experimental setup and consider optical conditions, including beam profile geometry and fluorescence pattern formation. A realistic formula that can explain the parameters governing the EMOFPAP method using our present optical setup has been derived. A comparison of results between experimental and computer simulation data is made, and an optimization of the EMOFPAP method is proposed.

DNA↗

PubMedDate not supplied

The sieving of rod-shaped viruses during agarose gel electrophoresis. I. Comparison with the sieving of spheres.

The sieving of rod-shaped viruses during agarose gel electrophoresis is quantitatively analyzed here with a previously proposed model [G. A. Griess et al. (1989) Biopolymers, 28, 1475-1484] that has one radius (PE) of the effective pore at each concentration of gel. By use of this model and an internal spherical size standard, a plot of electrophoretic mobility vs agarose percentage is converted to a plot of the radius of the effective sphere (effective radius) vs PE. Experimentally, when the concentration of the rod-shaped bacteriophage, fd, is progressively increased, eventually the electrophoretic mobility of fd becomes dependent on its concentration. The concentration of fd at which this occurs decreases as the agarose concentration decreases. After avoiding this dependence on the concentration of sample, the effective radius of rod-shaped particles, including bacteriophage fd, length variants of fd, and length variants of tobacco mosaic virus, is found to increase as PE increases until a plateau of approximately constant maximum effective radius is reached at PcE. In the region of this plateau, the effective sphere's measure that best approximates that of the rod is surface area. However, significant disagreement with the data exists for surface area; the maximum effective radius for fd varies as (length)0.69. For fd and its length variants, the value of 2.PcE/length increases from 0.21 to 0.86 as the length decreases from 2808 to 367 nm. The dependence of effective radius on PE and the proximity of 2.PcE to the length of the rod are explained by (a) random orientation of rods at PE values in the region of the plateau, and (b) increasingly preferential end-first orientation (reptation) of the rod as PE decreases below PcE. This hypothesis of reptation is supported by a significant dependence of electrophoretic mobility on electrical potential gradient for a PE below, but not above, PcE. The dependence of 2.PcE/length on length is not rigorously understood, but is qualitatively explained by flexibility of the rods. This apparent flexibility has thus far prevented determination of a rod's axial ratio from quantitation of sieving during agarose gel electrophoresis. The electrical potential dependence of electrophoretic mobility is determined here by a procedure of two-dimensional agarose gel electrophoresis. This procedure is also useful for detecting rod-shaped particles in heterogeneous mixtures of predominantly spherical particles.

Bacteriophages↗

PubMed1990

Gel electrophoresis of micron-sized particles: a problem and a solution.

The gel electrophoresis of spherical particles with a radius above 0.2 micron has not been reported yet. In the present study, video phase-contrast light microscopy is used to observe the motion of individual latex spheres, 0.52 micron in radius, during electrophoresis in 0.1% agarose gels. At 2 V/cm, the spheres initially migrate in the direction of the electrical field. However, each sphere eventually undergoes a cessation of all motion. Brownian motion is restored when the electrical potential gradient is reduced to zero. Arrest can be prevented by periodically inverting the direction of the electrical field. These observations are explained by electrical field-induced steric trapping of the spheres by gel fibers. Inversion of the electrical field should assist the application of agarose gel electrophoresis to micron-sized cellular organelles and cells.

Bacteria↗

PubMed1989

Protein electrophoresis of diagnostic peritoneal lavage in penetrating abdominal trauma: a marker of significant injury.

Protein electrophoresis of diagnostic peritoneal lavage fluid has been shown in experimental work to be a marker of hollow viscus injury. In order to ascertain the value of this investigation in penetrating abdominal trauma, we conducted a prospective study, over a 4-month period, involving 35 patients with stab wounds to the abdomen whose initial clinical examination was equivocal. The results of electrophoresis were compared with the standard quantitative criteria for diagnostic lavage and the operative findings. Protein electrophoresis was found to be a more sensitive and specific marker of significant intra-abdominal injury than the standard quantitative criteria.

Abdominal Injuries↗

PubMed1989

Determination of total and free concentration of propranolol in human plasma by displacement electrophoresis in a two-layer polyacrylamide gel using fluorimetric detection.

A new method based on displacement electrophoresis has been developed for the determination of the total and free concentration of propranolol, a beta-adrenergic blocker drug, in plasma. To determine the total concentration the drug is extracted from human plasma into a chloroform + heptane mixture in the presence of ammonia. After evaporation of the solvent mixture the residue is submitted to displacement electrophoresis in a glass tube containing a two-layer polyacrylamide gel. When the propranolol electrophoretically leaves the gel column it is transferred by a buffer flow to the cuvette of a fluorimeter for continuous detection and quantification. The concentration of the free non-protein bound drug can be determined by the same displacement electrophoresis technique following extraction of the plasma sample into the above organic solvent mixture in the absence of ammonia. Alternatively the extraction procedure can be exchanged for dialysis for 1 h. To decrease the risk that large size material might comigrate with propranolol two layers of polyacrylamide are used, one having small pores to retard such material. In addition, we use fluorimetric detection which means that many possible contaminants are not recorded and therefore do not affect a quantitative determination of propranolol.

Ammonia↗

PubMedDate not supplied

High-resolution separation of PCR product and gene diagnosis by capillary gel electrophoresis.

High-resolution separation of a PCR product from a mixture of DNA restriction fragments was achieved using capillary gel electrophoresis. The capillary gel electrophoretic separation gives an excellent resolution of two fragments of the 500-bp PCR product and the 506-bp DNA fragment, which differs by only 6 bp, and the complete separation of a broader chain length range of DNA afragments up to 12 kbp within 20 min. The plate number of gel-filled capillary was achieved to be 2 million plates per meter. Capillary gel electrophoresis is applied to the gene diagnosis for heart disease through apolipoprotein E genotyping. The advantages and limitations of capillary gel electrophoresis in the application to PCR analysis and DNA diagnosis are discussed.

Apolipoproteins E↗

PubMed1988

Rat liver cytosolic protein changes after ethanol exposure studied by two-dimensional electrophoresis.

Rats were fed liquid food containing ethanol in concentrations ranging from 1-5% for 13 weeks. Livers were removed for histopathology and the liver cytosolic protein fraction was prepared and used for two-dimensional gel electrophoresis (2D-PAGE). Polypeptides were visualized by silver staining. Scanning was made for estimation of the relative abundance of protein in each polypeptide spot in the gels and for comparison between rats. Visual inspection and scanning of gels with the stained polypeptide spots obtained after equilibrium isoelectric focusing and non-equilibrium pH gradient electrophoresis revealed that: 1) within the control rat and ethanol-treated rat livers the numbers of polypeptide spots detected using isoelectric focusing in the first dimension were approximately 500 and for non-equilibrium pH gradient electrophoresis 400; 2) in the control group the variation in the estimated amount of protein in each spot was remarkably small; 3) pronounced differences in the relative abundance of protein in several of the spots was observed in the ethanol-exposed rats as compared to controls. Dose-response relations and possible causes for the effects of ethanol are discussed.

Animals↗

PubMed1988

Horizontal two-dimensional electrophoresis with immobilized pH gradients using PhastSystem.

Protocols for horizontal two-dimensional electrophoresis with immobilized pH gradients in the first dimension were modified for horizontal micro two-dimensional electrophoresis using PhastSystem. Different equilibration conditions of the first-dimensional immobilized pH gradient gel strip prior to second-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evaluated. Silver stained two-dimensional patterns were obtained within 3.5 h.

Electrophoresis, Gel, Two-Dimensional↗

PubMed1988

Separation accuracy of free flow electrophoresis as proved by flow cytometry.

The separation accuracy of the free flow electrophoresis ACE 710 device was proved by immunological methods. Several cell populations of human peripheral blood were purified using physical cell isolation methods such as countercurrent centrifugal elutriation and cell electrophoresis. The purified cell fractions were treated with fluoresceinisothiocyanate-labelled antibodies. The percentages of cells in each fraction binding the antibodies of interest were determined by flow cytometry. The analyses revealed that with the help of free flow electrophoresis, given cell populations of human peripheral blood can be highly enriched and that preenrichment of minor cell populations enhances the efficacy of a flow cytometer.

Cell Separation↗

PubMed1988

A continuous acetic acid system for polyacrylamide gel electrophoresis of gliadins and other prolamines.

A polyacrylamide gel electrophoresis system buffered by acetic acid alone was developed for electrophoresis of prolamines. When applied to gliadin electrophoresis, the acetic acid system produces more bands than does a conventional aluminum lactate-lactic acid system (using 12% acrylamide gels). The acetic acid system is relatively simple, requiring a single buffer component that is universally available in high purity.

Acetates↗

PubMed1988

Physical identification of a virus in a crude leaf extract by its Ferguson plot in agarose gel electrophoresis.

Crude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto-)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus.

Buffers↗