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PubMed2003

Hybrid microdevice electrophoresis of peptides, proteins, DNA, viruses, and bacteria in various separation media, using UV-detection.

Recently we described the design of a hybrid microdevice for micro(nano)electrophoresis and electrochromatography, discussed its advantages and disadvantages compared to conventional microdevices and presented a few applications with low-molecular-weight samples. In this paper, we demonstrate the broad application range of this device using UV-based analyses of (i) peptides by free-zone electrophoresis and electrophoresis in a recently introduced gel (polyacrylamide cross-linked with allyl-beta-cyclodextrin), (ii) proteins by electrophoretic molecular-sieving in a polymer solution supplemented with SDS, (iii) DNA fragments by electrophoresis in the above gel, (iv) virus particles in this gel, as well as in free buffer and (v) bacteria in free buffer. To illustrate the advantages of the hybrid microdevice we can mention that electrophoresis of proteins in a polymer-containing buffer, supplemented with sodium dodecyl sulfate (SDS), in a 4.30 (2.75) cm long channel gave a resolution similar to that in conventional capillary electrophoresis in a 23.5 (18.6) cm long capillary and analysis times which were 15-fold shorter.

Bacteria↗

PubMed1981

Analysis of pulmonary surfactant apoproteins by electrophoresis.

Surfactant apoproteins were prepared from detergent-solubilized rat surfactant by immunoaffinity chromatography. SDS-polyacrylamide gel electrophoresis of the apoproteins, without prior chemical reduction, revealed several bands of molecular weights 50 000-78 000, 140 000 and 160 000. Following treatment with dithiothreitol, the apoproteins were resolved into three bands of molecular weights 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 daltghts 38 000, 32 000 and 26 000. Further analysis of the apoproteins by two-dimensional polyacrylamide gel electrophoresis showed that each of the proteins of molecular weights 38 000, 32 000 and 26 000 were crosslinked by disulfide bridges and formed homopolymers. Based on periodic acid-Schiff staining, the 38 000 dalton protein appeared to be the richest in carbohydrates, followed by the 32 000 and 26 000 dalton proteins. Partial proteolysis of the 38 000 and 32 000 dalton proteins showed similarity in the sizes of peptides generated. Surfactant-associated proteins from human and monkey lungs were also analyzed by polyacrylamide gel electrophoresis. A non-serum glycoprotein of molecular weight 38 000 was found. In different systems of polyacrylamide gel electrophoresis, this protein showed an electrophoretic mobility similar to that of the 38 000 dalton protein of rat surfactant. However, it formed polymers of molecular weight higher than those of polymers found in rat surfactant.

Animals↗

PubMed1995

Capillary zone electrophoresis of serum proteins: study of separation variables.

Electrophoresis of serum proteins is one of the traditional applications of zone electrophoresis. Whereas electrophoresis in supporting media uses usually 5,5'-diethylbarbiturate at pH 8.6 as the buffer, in capillary zone electrophoresis with on-line UV detection, this electrolyte is of little use because of its high UV absorbance. For that reason, a number of operational electrolytes differing in composition were tested for use in capillary electrophoresis of serum proteins. The influence of the pK(A) of co-ions and counter ions and the concentration of the operational electrolyte was examined. If 0.1 M methylglucamine-0.1 M epsilon-aminocaproic acid or 0.1 M methylglucamine, -0.1 M gamma-aminobutyric acid is used as the operational electrolyte, capillary electrophoresis separates serum proteins into more than ten zones.

Blood Proteins↗

PubMed1983

A study of cell electrophoresis as a means of purifying growth hormone secreting cells.

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partially purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flow electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.

Animals↗

PubMed1995

Capillary electrophoresis: a new tool in forensic toxicology. Applications and prospects in hair analysis for illicit drugs.

Capillary electrophoresis, the modern approach to instrumental electrophoresis, is probably the most rapidly expanding analytical technique that has appeared in recent years. In the hands of forensic toxicologists, capillary electrophoresis (CE) represents a powerful new analytical tool, which has proved suitable for the investigation of illicit drugs in seized preparations and also in complex biological matrices, among which is hair. CE can be applied according to different separation mechanisms, and among those that are toxicologically relevant are capillary zone electrophoresis and micellar electrokinetic capillary chromatography, which display different selectivities. For the investigation of hair for drugs of abuse, capillary electrophoresis proved effective, providing simultaneous determinations of different drugs without derivatization, with acceptable sensitivity (typically better than 1 ng of drug per mg of hair). The possibility of carrying out determinations of the same analytes, based on different separation mechanisms (capillary zone electrophoresis and micellar electrokinetic chromatography) with the same instrumentation, simply changing the buffer composition, provides an interesting possibility of 'internal' confirmation of the results.

Electrophoresis↗

PubMed1996

Capillary electrophoresis: principles and applications in illicit drug analysis.

Capillary electrophoresis, which appeared in the early 1980s, is now rapidly expanding into many scientific disciplines, including analytical chemistry, biotechnology and biomedical and pharmaceutical sciences. In capillary electrophoresis,electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus obtaining high efficiencies (N > 10(5)) and excellent mass sensitivities (down to 10(-18)-10(-20) moles). The main features of capillary electrophoresis are: versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, extremely low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems, ruggedness and simplicity of instrumentation. Capillary electrophoresis applications in forensic sciences have appeared only recently, but are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis, from both the instrumental and analytical points of view. Furthermore, the main applications in the analysis of illicit/controlled drugs in both illicit preparations and biological samples are presented and discussed (43 references). It is concluded that the particular separation mechanism and the high complementarity of this technique to chromatography makes capillary electrophoresis a new powerful tool of investigation in the hands of forensic toxicologists.

Electrophoresis, Capillary↗

PubMed2005

Highly sensitive determination of the methylated p16 gene in cancer patients by microchip electrophoresis.

The p16 tumor suppressor gene is inactivated by promoter region hypermethylation in many types of tumor. Recent studies showed that aberrant methylation of the p16 gene is an early event in many tumors, especially in lung cancer, and may constitute a new biomarker for early detection and monitoring of prevention trials. We detected tumor-associated aberrant hypermethylation of the p16 gene in plasma and tissue DNA from 153 specimens using a modified semi-nested methylation-specific PCR (MSP) combining plastic microchip electrophoresis or slab gel electrophoresis, respectively. Specimens were from 79 lung cancer patients, 15 abdominal tumor patients, 30 positive controls and 30 negative controls. The results showed that the positive rate obtained by microchip electrophoresis was more than 26.6% higher and the same specificity was kept when compared with slab gel electrophoresis. The microchip electrophoresis can rapidly and accurately analyze the PCR products of methylated DNA and obviously improve the positive rate of diagnosis of cancer patients when compared with gel electrophoresis. This method with the high assay sensitivity might be used for detection of methylation of p16 gene and even to facilitate early diagnosis of cancer patients.

Abdominal Neoplasms↗

PubMed1999

The influence of temperature during alkaline treatment and electrophoresis on results obtained with the comet assay.

The alkaline comet assay (single-cell gel electrophoresis) is becoming established as a genotoxicity test with many fold applications in vitro and in vivo. While the underlying principles are identical, various modifications of the method are in use which clearly affect the sensitivity and resolving power of the assay. One variable of potential importance that has been disregarded until now is temperature during alkaline treatment and electrophoresis. We therefore performed comet assay experiments with human blood and V79 Chinese hamster cells using two different temperatures (4 and 20 degrees C, i.e. room temperature) during alkaline treatment and electrophoresis. DNA damage was induced by the two standard mutagens gamma irradiation and methyl methanesulfonate (MMS). The results clearly indicate significant differences in the detection of background and mutagen-induced DNA damage at these two temperatures. Under otherwise identical test conditions (including the duration of alkaline treatment and electrophoresis), increased temperature during alkaline treatment and electrophoresis strongly enhances DNA migration. Our findings suggest that the comet assay should be performed under strictly controlled and reproducible temperature conditions. In any case the temperature during alkaline treatment and electrophoresis should be stated in a publication to allow for a critical evaluation of results obtained with the comet assay.

Alkalies↗

PubMed2002

Effectiveness of capillary electrophoresis using fluorescent-labeled primers in detecting T-cell receptor gamma gene rearrangements.

We describe the use of fluorescent-labeled primers to analyze T-cell receptor gamma gene rearrangements (TCR gamma GR) using capillary electrophoresis in the ABI Prism 310 Genetic Analyzer. We also compare the performance with denaturing gradient gel electrophoresis (DGGE). In a single multiplex polymerase chain reaction (PCR) we amplified TCR gamma GR with primers for all known groups of variable region genes, and joining region genes described in lymphoid neoplasms. Ten reactive samples, followed by five cell lines and 25 tumor samples with 41 individual TCR gamma GR (due to many biallelic rearrangements) previously identified by DGGE, were analyzed to validate the technique. The capillary electrophoresis protocol has 92% concordance for both TCR clonal status (23 of 25) and 95% concordance in the number of individual TCR gamma GR (38 of 41) identified by DGGE. The reproducible sensitivity for detecting TCR gamma GR diluted in reactive lymphoid DNA is 2% in clinical applications. Discrimination of predominant rearrangements requires a minimum ratio of two times the height of the normal distribution of polyclonal peaks. Capillary electrophoresis can provide results within 60 minutes for each specimen after PCR is complete. Capillary electrophoresis provides a faster result than sequence-based separation methods and gives an archival electronic record. Fluorescent labeling allows the identification of both the variable and joining gene segments used in a TCR gamma GR. The effectiveness of capillary electrophoresis is similar to DGGE.

Base Sequence↗

PubMed2007

Instrumentation for medium-throughput two-dimensional capillary electrophoresis with laser-induced fluorescence detection.

In two-dimensional capillary electrophoresis, a sample undergoes separation in the first dimension capillary by sieving electrophoresis. Fractions are periodically transferred across an interface into a second dimension capillary, where components are further resolved by micellar electrokinetic capillary electrophoresis. Previous instruments employed one pair of capillaries to analyze a single sample. We now report a multiplexed system that allows separation of five samples in parallel. Samples are injected into five first-dimension capillaries, fractions are transferred across an interface to 5 second-dimension capillaries, and analyte is detected by laser-induced fluorescence in a five-capillary sheath-flow cuvette. The instrument produces detection limits of 940 +/- 350 yoctomoles for 3-(2-furoyl)quinoline-2-carboxaldehyde labeled trypsin inhibitor in one-dimensional separation; detection limits degrade by a factor of 3.8 for two-dimensional separations. Two-dimensional capillary electrophoresis expression fingerprints were obtained from homogenates prepared from a lung cancer (A549) cell line, on the basis of capillary sieving electrophoresis (CSE) and micellar electrophoresis capillary chromatography (MECC). An average of 131 spots is resolved with signal-to-noise greater than 10. A Gaussian surface was fit to a set of 20 spots in each electropherogram. The mean spot width, expressed as standard deviation of the Gaussian function, was 2.3 +/- 0.7 transfers in the CSE dimension and 0.46 +/- 0.25 s in the MECC dimension. The standard deviation in spot position was 1.8 +/- 1.2 transfers in the CSE dimension and 0.88 +/- 0.55 s in the MECC dimension. Spot capacity was 300.

Biogenic Amines↗

PubMed1986

Densitometric scanning of high-resolution electrophoresis of serum: methodology and clinical application.

The recent introduction of high-resolution electrophoresis into many clinical laboratories has expanded the information available about protein abnormalities. While most laboratories interpret high-resolution electrophoresis patterns by direct visual examination of the stained electrophoresis strips, much useful information can be attained by supplemental densitometric scanning of these preparations. In the present report, we detail a method to perform densitometric scanning of high-resolution electrophoresis strips, and compare these results to those performed by standard cellulose acetate electrophoresis. Further, we evaluate the quantitative information by comparing the results from the densitometer scans with nephelometric quantification of specific monoclonal proteins. The present method gives a correlation coefficient of 0.97 when the gamma region densitometric scan is compared to the quantification of IgG by nephelometry in patients with known gamma-migrating IgG monoclonal gammopathies. Further, twofold dilution studies of these specimens showed excellent linearity. By providing objective, reliable, quantitative information, densitometric scanning of high-resolution electrophoresis strips is a useful adjunct to direct visual examination of these specimens.

Blood Protein Electrophoresis↗

PubMed1983

Analysis of apolipoproteins B100 and B48 by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis.

Apolipoproteins B100 and B48 in human and rat plasma were studied by using sodium dodecyl sulfate (SDS) polyacrylamide gradient gel electrophoresis. On SDS gradient gel electrophoresis, human and rat apoprotein B100 co-migrated and had an apparent Mr equal 258,000 +/- 12,000. Human and rat apoprotein B48 had an apparent Mr equal 189,000 +/- 6,000. The molecular weight of human apoprotein B100 determined by sedimentation equilibrium analysis was 270,000 +/- 20,000, which was similar to the value determined by SDS gradient gel electrophoresis. However, on SDS polyacrylamide gel electrophoresis at constant concentration, the relative migration value of human apoprotein B100 was not constant when the concentration of polyacrylamide was changed. These results indicate that SDS gradient gel electrophoresis is more suitable for the analysis of apolipoprotein B's than ordinary SDS polyacrylamide gel electrophoresis.

Animals↗

PubMed2000

Capillary electrophoresis: a new analytical tool for forensic toxicologists.

In capillary electrophoresis, electrophoretic or electrokinetic separations are carried out in tiny capillaries at high voltages (10-30 kV), thus achieving high efficiency (N > 105), resolution power, and mass sensitivity (down to 10(-18)-10(-20) moles). The main characteristics of capillary electrophoresis are versatility of application (from inorganic ions to large DNA fragments), use of different separation modes with different selectivity, low demands on sample volume, negligible running costs, possibility of interfacing with different detection systems including mass spectrometry, and the ruggedness and simplicity of the instrumentation. Capillary electrophoresis applications in the forensic sciences are now rapidly growing, particularly in forensic toxicology. The present paper briefly describes the basic principles of capillary electrophoresis and presents a selected review of its main applications to the analysis of illicit/controlled drugs in biologic samples. An original analytical approach to the determination of carbohydrate deficient transferrin, a new marker of chronic alcohol abuse, based on capillary electrophoresis is also described. It is concluded that the peculiar separation mechanisms and the high complementarity of capillary electrophoresis to chromatography make it a new powerful tool of investigation in the hands of forensic toxicologists.

Electrophoresis, Capillary↗

PubMed1985

A simple routine method for SDS-electrophoresis of urinary proteins in kidney transplant patients.

The molecular weight analysis of urinary proteins can provide useful diagnostic information. For this purpose a routine method of sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-electrophoresis) is described. The main problem in the introduction of this method into the clinical laboratory lies in the availability of "ready-to-use" electrophoresis gels with good and reproducible quality. A device for gel production is therefore described, which is easily constructed from plastic package materials. Polyacrylamide gels are made batch-wise and in advance. They are suitable for various types and techniques of horizontal electrophoresis. Further, a method for analysis of urinary proteins is described, which permits the simultaneous analysis of 22 unconcentrated urine samples within a 3-hour-electrophoresis run. A specially optimized Coomassie blue staining method (overnight) ensures detection of proteins in the concentration range of milligrams per litre. Electrophoretic analyses were documented by photocopying the finished electrophoresis gels.

Electrophoresis, Polyacrylamide Gel↗

PubMed1999

Electrophoresis in space.

Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface.

Animals↗

PubMed2001

[Capillary array electrophoresis and high-throughput DNA sequencing].

Depending on the outputs of 10,080 DNA sequencing samples from the human genome project, technical features of capillary array electrophoresis are discussed. The results show that 88% of the total readlength were higher than 50 kbp/run. It means that most of the readlength could reach 500 bp or more with reasonable quality. The results were much better than expected when the technology of capillary array appeared about one year ago. A key parameter affecting the quality of DNA sequencing is the purity of sequencing template. Therefore, a batch agarose slab electrophoresis was applied to check the purity before loading. Other conditions, such as concentration of template and loading amount, are discussed as well. Finally, comparisons based on the electrophoresis timing, the pre-treatment of samples, the loading procedures and the results analysis between capillary array electrophoresis and slab gel electrophoresis have been made. From the point of view of high-throughput DNA sequencing, especially in the field of genome research, the capillary array electrophoresis should be the better choice as a more efficient technical platform.

Base Sequence↗

PubMed1987

Electrophoresis and the isomune-LD and LD-1 immuno methods compared for measurement of lactate dehydrogenase isoenzyme-1.

We evaluated three different methods for measuring lactate dehydrogenase (EC 1.1.1.27) isoenzyme LD-1 activity--agarose gel electrophoresis and two immunoassays, Isomune-LD (Roche) and LD-1 Immuno (Seragen)--in patients' samples for which measurement of creatine kinase-MB was ordered. Regression analyses of the comparisons gave the following results: LD-1 (%) from Isomune-LD (y) vs electrophoresis (x) (n = 51), y = 1.05x + 1.99, r = 0.92; LD-1 (%) from LD-1 Immuno (y) vs electrophoresis (n = 27), y = 1.05x + 3.94, r = 0.88; LD-1 (%) from LD-1 Immuno (y) vs Isomune-LD (x) (n = 41), y = 1.06x + 0.48, r = 0.95. Comparison by Student's paired t-test yielded significant differences between the mean values by electrophoresis and both Isomune-LD (P less than 0.005) and LD-1 Immuno (P less than 0.001), but no significant difference between the two immunoassays (P greater than 0.2). Analyzing these results by the overlap index, we conclude that electrophoresis shows the best clinical correlation followed, in order, by the Isomune-LD and the LD-1 Immuno methods. Both immunoassays are simpler and more rapid than electrophoresis, but in our hands the Isomune-LD method demonstrated greater precision and better correlation with electrophoretic values.

Electrophoresis↗

PubMed1998

Application of free-flow electrophoresis to the purification of trichosanthin from a crude product of acetone fractional precipitation.

The application of free-flow electrophoresis (FFE) to the purification of trichosanthin (TCS) from a crude product of acetone fractional precipitation was investigated. An electrophoresis technique, combining field step electrophoresis (FSE) and zone electrophoresis (ZE) to a one-step procedure, was optimized until a satisfactory purification factor (1.35), high resolution, and purity (>99%) were achieved. Testing several separation buffer systems revealed that a throughput of 14.2 mg/h can be obtained when the very basic TCS (pI 10.1) was dissolved and electrophoresed in a phosphate buffer system of pH 4. The purity of electrophoresed trichosanthin was proved by a variety of analytical methods, such as sodium dodecyl sulfate (SDS)-gel electrophoresis, capillary isoelectric focusing (CIEF), and sequencing of N- and C-termini. The high purity and large throughput achieved at low cost by using FFE indicates that this method can be employed for TCS purification.

Acetone↗