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PubMed1997

Microgel electrophoresis: sensitivity, mechanisms, and DNA electrostretching.

Based on the treatment of microgels to remove proteins, we speculate that proteins may be bound to DNA in the microgels even after electrophoresis. We speculate that some DNA single-strand breaks may be a reflection of these protein-DNA complexes. We suggest methods to limit such artifacts, and present data demonstrating a lymphocyte DNA double-strand break sensitivity of 12.5 rads and day-to-day reproducibility of microgel electrophoresis using these principles. Extending these principles, we describe DNA behavior during alkaline and neutral microgel electrophoresis based on observations of the stained DNA and its migration patterns. During microgel electrophoresis, individual DNA molecules behave as if anchored at one end while the other end is free to migrate in response to the electric field. We capitalize on this behavior by developing a neutral microgel method to stretch chromosomes.

DNA Damage↗

PubMedDate not supplied

Gel electrophoresis-autoradiographic image analysis of radiolabeled protein drug concentration in serum for pharmacokinetic studies.

INTRODUCTION: The purpose of this study was to evaluate the feasibility of using gel electrophoresis combined with autoradiographic image analysis for quantitating protein drug concentrations in biological fluid for pharmacokinetic studies. METHODS: Protein drugs were iodinated using the Iodogen reagent and injected into Sprague-Dawley rats for pharmacokinetic evaluation. Serum samples were analyzed using trichloroacetic acid (TCA)-precipitable counts or sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Commercially available precasted Bis-Tris gradient gels were used for SDS-PAGE. Autoradiography of gel samples was performed using phosphoimager and quantitated using the ImageQuant software. RESULTS: The maximum loading volume for protein drugs with molecular weight close to that of albumin ( approximately 70 kDa) was about 1 microl, whereas for protein drugs with larger or smaller molecular weight (i.e., >80 or <40 kDa), the maximum loading volume was up to 20 microl/lane. The optimal exposure time was about 18 h or overnight. Standard curves were constructed using serially diluted dosing solution, which was linear over a 10-fold concentration range with a correlation coefficient of.98. Comparing to the soluble human interleukin-13 receptor (shIL-13R) pharmacokinetic profiles from TCA-precipitable counts, the quantitative gel analysis revealed lower concentrations at later time points and a lower bioavailability from intraperitoneal injection. DISCUSSION: This study provided the first systemic evaluation of gel electrophoresis technology for quantitative protein drug determination in serum and its application in pharmacokinetic studies. The combination of gel electrophoresis with autoradiographic image analysis provided accurate and specific quantitation results. The overnight turnover time allowed routine application in preclinical pharmacokinetic studies.

Animals↗

PubMed1999

An updated two-dimensional gel electrophoresis technique for the detection of drug-induced changes in protein phosphorylation in intact smooth muscle.

Two-dimensional gel electrophoresis is widely used in many areas of scientific research. The necessity for greater resolution and more sensitive protein detection with this technique have resulted in a steadily changing methodology. Complete descriptions of some aspects of two-dimensional gel electrophoresis are available in the earlier literature. However, simplified methods incorporating recent advances specifically designed to use two-dimensional gel electrophoresis for the measurement of protein phosphorylation in intact tissue are lacking. This report describes, in detail, each of the steps involved in carrying out such measurements including intact tissue labeling with 32P, homogenization and protein sample preparation, two-dimensional gel electrophoresis using isoelectric focusing followed by vertical second-dimension SDS-PAGE, staining, autoradiography, and quantitative analysis of changes in phosphorylation of specific proteins. This method incorporates a number of modifications taken from other published sources and includes several novel changes developed in our laboratory. To illustrate the utility of this technique we have included a set of results analyzing the phosphorylation patterns induced by the addition of a nitric oxide donor, sodium nitroprusside, to intact strips of rat aorta. We were able to demonstrate SNP-induced phosphorylation of a number of proteins, several of which have not been previously described in earlier reports in which the patterns of PKG-mediated phosphorylation were investigated.

Animals↗

PubMed1991

Separation of DNA restriction fragments by capillary electrophoresis using coated fused silica capillaries.

The abilities of several different capillary electrophoresis techniques to separate DNA restriction fragments up to 23,000 bp were investigated. Methods employing electroosmotic flow in an untreated silica capillary were found to provide, at best, only partial resolution of the 23 fragments in a 1-kbp DNA ladder. By coating the inner walls of a silica capillary with poly(acrylamide) and filling these capillaries with buffers containing methylcellulose as a sleving medium, all fragments in the 1-kbp DNA ladder were separated. In addition, this technique facilitated the separation of the very large fragments in a lambda DNA-HindIII digest. Optimum resolution was obtained at low separation potentials using buffers containing at least 0.5% methylcellulose. The performance of this technique, i.e., resolution and quantitation, make capillary electrophoresis a powerful complement to slab gel electrophoresis and may make it a preferred alternative to both agarose gel electrophoresis and HPLC for applications such as the confirmation of plasmid integrity.

DNA↗

PubMed1994

Postcolumn radionuclide detection of low-energy beta emitters in capillary electrophoresis.

A postcolumn radionuclide detection system for capillary electrophoresis (CE) is described. Eluant from an electrophoresis capillary is directed onto a peptide binding membrane that has been previously coated with a solid scintillator. The membrane is moved in a preselected pattern relative to the fixed capillary outlet during electrophoresis. Light emission from scintillation is imaged onto a charge-coupled device (CCD) using a series of 35-mm camera lenses. Detection of two low-energy beta- emitters (35S and 3H) not previously reported for capillary electrophoresis is demonstrated. The separation efficiencies are similar to those obtained with on-line UV detection. The response for 35S-labeled methionine is linear (r2 = 0.996) from 66 amol to 11 fmol. Detection limits are 88 zmol (0.03 Bq) for 32P-labeled analytes, 17 amol (0.94 Bq) for 35S-labeled analytes, and 8 fmol (8.5 Bq) for 3H-labeled analytes.

Beta Particles↗

PubMed2001

Molar mass profiling of synthetic polymers by free-solution capillary electrophoresis of DNA-polymer conjugates.

The molar mass distribution of a polymer sample is a critical determinant of its material properties and is generally analyzed by gel permeation chromatography or more recently, by MALDI-TOF mass spectrometry. We describe here a novel method for the determination of the degree of polymerization of polydisperse, uncharged, water-soluble polymers (e.g., poly(ethylene glycol) (PEG)), based upon single-monomer resolution of DNA-polymer conjugates by free-solution capillary electrophoresis. This is accomplished by end-on covalent conjugation of a polydisperse, uncharged polymer sample (PEG) to a monodisperse, fluorescently labeled DNA oligomer, followed by electrophoretic analysis. The monodisperse, charged DNA "engine" confers to each conjugate an equal amount of electromotive force, while the varying contour lengths of the uncharged, polydisperse polymers engender different amounts of hydrodynamic drag. The balance of electromotive and hydrodynamic forces enables rapid, high-resolution separation of the DNA-polymer conjugates as a function of the size of the uncharged PEG tail. This provides a profile of the molar mass distribution of the original polymer sample that can be detected by laser-induced fluorescence through excitation of the dye-labeled DNA. We call this method free solution conjugate electrophoresis (FSCE). Theory-based analysis of the resulting electrophoresis data allows precise calculation of the degree of polymerization of the PEG portion of each conjugate molecule. Knowledge of the molecular mass of the uncharged polymer's repeat unit allows for direct calculation of the molar mass averages as well as sample polydispersity index. The results of these analyses are strikingly reminiscent of MALDI-TOF spectra taken of the same PEG samples. PEG samples of 3.4-, 5-, and 20-kDa nominal average molar mass were analyzed by FSCE and MALDI-TOF; the values of the molar mass averages, Mw and Mn, typically agree to within 5%. Measurements and molar mass calculations are performed without any internal standards or calibration. Moreover, when DNA-polymer conjugate analysis is performed in a chip-based electrophoresis system, separation is complete in less than 13 min. FSCE offers an alternative to MALDI-TOF for the characterization of uncharged, water-soluble polymers that can be uniquely conjugated to DNA.

DNA↗

PubMed1990

Capillary electrophoresis in 2 and 5 microns diameter capillaries: application to cytoplasmic analysis.

Capillary electrophoresis with electrochemical detection in 2- and 5-micron capillaries has been developed to study ultrasmall biological environments. Sample volumes as low as 270 fL have been injected into the electrophoresis capillary with subattomole detection limits for easily oxidized species. We have applied this method to the analysis of single cell cytoplasm. Sampling of the cytoplasm is accomplished by inserting one end of the electrophoresis capillary directly into a single nerve cell. The high-voltage end of the electrophoresis capillary has been etched with hydrofluoric acid to form a microinjector. This injection scheme represents an improvement over those previously used for similar applications. The excellent selectivity of this method is demonstrated for catecholamine and indolamine neurotransmitters and their metabolites found in the invertebrate system, the pond snail Planorbis corneus.

Animals↗

PubMed2002

Capillary electrophoresis of cytochrome P-450 epoxygenase metabolites of arachidonic acid. 2. Resolution of stereoisomers.

Each of the four regioisomers of epoxyeicosatrienoic acids (EETs) is a candidate for being an endothelial-dependent hyperpolarizing factor (EDHF). One regioisomer, 14,15-EET, stereospecifically blocks cyclooxygenases from converting arachidonic acid to prostaglandins and stereospecifically binds to cellular receptors. Both stereospecific actions emphasize the need to establish the tissue availability of the 14,15-EET enantiomers. The present work describes a method to quantitate picogram amounts of 14,15-EET enantiomers by capillary electrophoresis. The 14,15-EET enantiomers were baseline resolved (R = 1.3) using unsubstituted beta-cyclodextrin and 32% acetonitrile (v/v). When absorption at 194 nm was monitored using a photodiode array detector, 8 and 1 pg of underivatized 14,15-EET were readily quantitated and detected, respectively. Capillary electrophoresis accurately assessed chiral excesses up to 97:3 for either 14,15-EET enantiomer. Moreover, capillary electrophoresis with a photodiode array detector was sufficiently sensitive to detect and measure 14,15-EET enantiomers from murine liver. Thus, unlike chiral-phase high-performance liquid chromatography, capillary electrophoresis can be used to directly assess the chirality of trace amounts of underivatized eicosanoids.

8,11,14-Eicosatrienoic Acid↗

PubMed2003

Tandem isotachophoresis-zone electrophoresis via base-mediated destacking for increased detection sensitivity in microfluidic systems.

Electrophoresis in microfluidic devices is becoming a useful analytical platform for a variety of biological assays. In this report, we present a method that allows for an increased sensitivity of detection of fluorescent molecules in microfluidic electrophoresis devices. This capability is provided by the implementation of a particular buffer system that is designed to initially function in an isotachophoretic (ITP) mode and, then after a controlled amount of electric current has been applied to the system, to transition to a zone electrophoretic mode. In the initial ITP mode, analytes dissolved in a large volume of injected sample are concentrated into a single narrow zone. After application of a sufficient and adjustable amount of electric current, the system switches into a zone electrophoretic mode, where the concentrated analytes are separated according to their electrophoretic mobilities. Application of this tandem ITP-zone electrophoretic strategy to the concentration, separation, and detection of fluorescent reporter molecules in a standard microfluidic device results in an approximately 50-fold increase in detection sensitivity relative to equivalent separations that are obtained with zone electrophoresis alone. Even with very long initial sample plugs (up to 3000 microm), this strategy produces electrophoretic separations with high resolutions and peak efficiencies. This strategy can be implemented to increase detection sensitivity in any standard microfluidic electrophoresis platform and does not require any specialized hardware or microchannel configurations.

Buffers↗

PubMed2003

Effect of dextran as a run buffer additive in drug-protein binding studies using capillary electrophoresis frontal analysis.

The study of drug-protein interactions by capillary electrophoresis frontal analysis requires establishment of a sufficient mobility difference between the mobility of the ligand and protein. The potential utility of dextran as a run buffer additive to manipulate the electrophoretic mobilities of low molecular weight ligands and protein in capillary electrophoresis frontal analysis binding studies was assessed. It was demonstrated that dextran was effective in improving the separation between the ligands warfarin and flurbiprofen and human serum albumin. Separation of ligand and protein increased with the concentration of added dextran (0-7.5% (w/w)), while molecular weight of the additive (70,000-2,000,000) only had a minor effect. The effect of dextran addition on viscosity and electrophoretic and electroosmotic mobilites was systematically studied. Optimal frontal analysis settings were a compromise between achieving satisfactory separation and acceptable analysis times without loss of plateau peak conditions. No effect of dextran upon the drug-human serum albumin interactions could be detected for the model ligands. Introduction of dextran into the electrophoresis buffer expands the applicability of capillary electrophoresis frontal analysis in drug research to binding interactions between proteins and low molecular weight ligands possessing similar electrophoretic mobilities.

Buffers↗

PubMed1999

Characterization of proteins from human cerebrospinal fluid by a combination of preparative two-dimensional liquid-phase electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

To purify and characterize low-abundance proteins in complex biological mixtures, we used a novel strategy that combined preparative two-dimensional liquid-phase electrophoresis (2D-LPE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Preparative 2D-LPE is based on the same isoelectric focusing and gel electrophoresis principles as the widely used analytical 2D gel electrophoresis, except that analytes remain in solution throughout separation. This novel approach shows many improvements compared to analytical 2D gel electrophoresis for the separation of proteins in biological fluids. For example, larger volumes/amounts of samples can be loaded, yielding sufficient amounts of low-abundance proteins for further characterization. Since proteins remain in liquid phase during the entire procedure, extra steps such as electroelution, extraction, or transfer to membranes from the gels prior to mass spectrometric analysis are obviated. We report the usefulness of 2D-LPE combined with MALDI-TOF MS for the purification and characterization of cystatin C and beta-2 microglobulin in human cerebrospinal fluid. This method should be applicable to a wide range of biological fluids, such as cerebrospinal fluid, serum, tissue extracts, cell media, whole cells, and bacterial lysates.

Cerebrospinal Fluid Proteins↗

PubMed2000

Automated ultra-thin-layer SDS gel electrophoresis of proteins using noncovalent fluorescent labeling.

Ultra-thin-layer SDS gel electrophoresis in conjunction with automated laser-induced fluorescence detection is a novel and powerful method for the analysis of fluorophore-labeled proteins. The technique described in this paper employs instant, noncovalent fluorophore labeling by the addition of a fluorescent staining dye to the sample proteins either during or immediately prior to the sample loading process. Thus, the method does not require time-consuming post- or preseparation staining/labeling. By combining the multilane format of SDS polyacrylamide slab gel electrophoresis and the high separation efficiency of capillary SDS gel electrophoresis, ultra-thin-layer SDS gel electrophoresis features rapid, high-throughput, and high-resolution analysis of proteins in the molecular mass range of 14-116 kDa. The good heat dissipation inherent to the ultrathin format enables the use of agarose and agarose-based composite separation matrixes, which can be easily replaced within the separation platform. Labeling efficiency as a function of the concentration of the staining dye, SDS, and proteins is thoroughly discussed. Detection sensitivity of the method was found to be at the low-femtomole level (1.25 ng/band), determined by analyzing a set of serial dilutions of standard proteins. Practical example of molecular mass determination and characterization of a complex protein mixture are also shown.

Autoanalysis↗

PubMed1989

Atypical sieving of open circular DNA during pulsed field agarose gel electrophoresis.

Pulsed field agarose gel (PFG) electrophoresis, originally used to improve the resolution by length of linear DNA [Cantor et al. (1988) Annu. Rev. Biophys. Biophys. Chem. 17, 287-304], is found here to cause atypical sieving of 48.5-97.0-kb open circular DNA. Two procedures of PFG electrophoresis are used: rotating gel electrophoresis with rotation of 2 pi radians [2 pi RGE; Serwer, P., & Hayes, S.J. (1989) Appl. Theor. Electrophor. (in press)] and field inversion gel electrophoresis [FIGE; Carle, G.F., Frank, M., & Olson, M. V. (1986) Science 232, 65-68]. During 2 pi RGE at 6 V/cm, the electrophoretic mobility (mu) of 48.5-kb open circular DNA increases in magnitude as agarose percentage (A) increases from 0.4 to 1.5. The sieving revealed by this mu vs A relationship is highly atypical (possibly unique) for any particle. The extent of this atypical sieving increases as electrical potential gradient, DNA length, and pulse time increase. In some cases a maximum is observed in a plot of mu's magnitude vs A. The mu of open circular lambda DNA is smaller in magnitude than the mu of equally long linear lambda DNA. Atypical sieving has also been observed by use of FIGE. As pulse times used during FIGE decrease below those achievable by 2 pi RGE, the progressive loss of circular DNA's atypical sieving is accompanied by both a dramatic increase in mu's magnitude at the lower A values and a decrease in mu's magnitude at the higher A values. At the lower A values, open circular DNA sometimes migrates more rapidly than linear DNA of the same length.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage lambda↗

PubMed1976

Two-dimensional gel electrophoresis of membrane proteins.

A high-resolution method for two-dimensional separation of membrane proteins is described. It involves a nondiscriminating solubilization of a membrane preparation with sodium dodecyl sulfate, followed by electrophoresis in the first dimension according to charge (by isoelectric focusing). The electrophoresis in the second dimension is in the presence of sodium dodecyl sulfate, thus separating proteins on the basis of molecular weight. Electrophoresis in the first dimension is either on a thin slab gel, or on a small-diameter tube; electrophoresis in the second dimension is on a thin slab gel. Up to 100 mug of protein can be analyzed. The two-dimensional system is a modification of the one recently described by O'Farrell (1975). About 150 different proteins can be visualized in Escherichia coli or Salmonella typhimurim cell envelopes; examples of differences between mutant and wild-type strains are presented. The method is applicable also to membrane preparations from other sources: a two-dimensional separation of plasma membrane proteins from HeLa cells is presented.

Bacterial Proteins↗

PubMed2005

Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

Diamond↗

PubMed2003

A new approach for screening for Y microdeletions: capillary electrophoresis combined with fluorescent multiplex PCR.

PURPOSE: To apply capillary electrophoresis for rapid screening for Y microdeletions METHODS: A set of 25 specific sequence tagged sites that cover the azoospermia factor a, b, and c regions of the Y chromosome was amplified in 5 fluorescent multiplex sets each including 5 primer pairs. One of the primers of each pair was labeled with a fluorescent tag attached to the 5'-end. After PCR amplification, analysis of the obtained PCR products was performed using capillary electrophoresis (ABI Prism 3100 Genetic Analyzer). The method was employed to determine Y microdeletions in azoospermic (n = 49) and severe oligozoospermic (n = 149) men. RESULTS: The number of PCR cycle (from 45 to 30) and the amount of DNA template (20-fold) used in fluorescent multiplex PCR were reduced because of the high sensitivity of capillary electrophoresis. Approximately 1000 multiplex PCR sets from 198 patients were analyzed simultaneously within 50 h. Y microdeletions were found in 3 out of the 198 azoospermic or severe oligozoospermic men. CONCLUSIONS: Application of capillary electrophoresis for detection of PCR products provides a semiautomated, high throughput method for rapid screening for microdeletions on the Y chromosome.

Chromosome Deletion↗

PubMed1980

Genetic heterozygosity in a natural population of Mus musculus assessed using two-dimensional electrophoresis.

Since the introduction of gel electrophoresis to population genetics, estimates of genic heterozygosity at allozyme loci have been made for many organisms. However, attempts to extend the range of proteins surveyed have been hampered by concern that there may have been bias in the loci sampled: nearly all proteins for which population data are available are soluble in low-salt extracts and most are enzymes belonging to particular groups such as the nonspecific esterases and phosphatases, various dehydrogenases and the enzymes of the glycolytic pathways and the citric acid cycle. Until recently, there were no techniques for estimating protein variation which were without such bias. Two-dimensional gel electrophoresis, however, offered hope of overcoming this bias, and it has proved useful for the resolution of proteins in crude homogenates and for estimating genetic variation. We have examined heterozygosity in a wild population of the house mouse (Mus musculus) by means of two-dimensional electrophoresis of whole kidney. As we report here, the observed level (2%) is substantially below the level detected by starch gel electrophoresis. Our results corroborate similar differences observed in Drosophila and man.

Animal Population Groups↗

PubMed1989

Conformational dynamics of individual DNA molecules during gel electrophoresis.

Gel electrophoresis is widely used in molecular biology to separate DNA molecules according to their sizes. The physical basis of this size separation is, however, poorly understood. Here we report observations of individual, fluorescently stained DNA molecules as they migrate during various kinds of gel electrophoresis. Their movement, under the influence of either a steady electric field or a pulsed-field, is characterized by cycles of elongation and contraction. Initially relaxed coils of DNA lengthen into 'hook-shaped' configurations which temporarily 'hang-up' on obstacles in the gel matrix before sliding off, contracting and entering another cycle. The effects of a new electrophoresis technique, termed 'pulse-oriented electrophoresis', which allows the effective angle of the electric field, and hence the molecular orientation of DNA, to be varied without electrode rearrangement, are also studied. In this case the DNA adopts a 'staircase' configuration showing that the net orientation in a direction is given by the vector sum of the pulses used.

DNA↗