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PubMed1994

Poly(ethylene glycol)methacrylate-acrylamide copolymer media for hydrophobic protein and low temperature electrophoresis.

The properties of poly(ethylene glycol)methacrylate-acrylamide copolymer media (PEGMACs) were exploited in two ways: (i) in the first-dimensional gel of two-dimensional electrophoresis (2-DE) of hydrophobic proteins and (ii) for high speed, high-resolution electrophoresis at low temperatures. In the first application, improved resolution and yield of isoelectric focusing (IEF) separations for the hydrophobic protein zein was achieved compared to IEF in standard polyacrylamide gels. This appears promising as a candidate approach for higher resolution 2-DE mapping of uncharacterized hydrophobic proteins. In the second application, PEGMACs compatible with hydroorganic antifreeze buffer systems below cooling of the gels to low temperatures (-20 degrees C), which allowed greater current to be tolerated during electrophoresis. PEGMAC gels enabled us to perform sixfold faster electrophoretic separations and achieve threefold improved resolution of six standard proteins at the lower temperatures in a direct comparison with the normal sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system. If this approach is coupled with more precise instrumentation to control low temperatures during electrophoresis, greater separation speeds and resolution may be anticipated.

Acrylamides↗

PubMed1995

Sodium chloride in separation medium enhances cell compatibility of free flow electrophoresis.

Free flow electrophoresis of cell suspensions in buffers containing sodium chloride was investigated using a modified procedure and the new apparatus Octopus PZE. The major methodical innovations are upward fluid flow, margin buffers flowing through the electrophoresis chamber at both sides of a central cell suspension buffer, adjacent to the electrode membranes, and a sample injection device which focuses the cells hydrodynamically to the middle of the chamber thickness. Mononuclear leukocytes, suspended in a buffer containing 35 mM NaCl, could be fractionated with the same accuracy as by conventional free flow electrophoresis, operated with a single NaCl-free chamber buffer. However, testing the vitality of separated cells with the help of the calcium indicator FURA2-AM clearly demonstrated the biological importance of the presence of a minimum amount of sodium chloride during cell electrophoresis. Only if at least 35 mM NaCl were present could an undisturbed cytosolic Ca2+ metabolism be maintained for the time of a free flow electrophoresis cell separation experiment.

B-Lymphocytes↗

PubMed1996

The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis.

DNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1-1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345-349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp)/D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time-dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings-Weiss model.

DNA↗

PubMed1996

Analysis of RNA by capillary electrophoresis.

Analytical parameters known to be important for the separation of DNA by capillary electrophoresis, including gel polymer concentration, electrical field strength and temperature, were investigated and optimized for the analysis of RNA molecules from 100 to 2000 bases. Denaturation, essential to obtain uniform and identifiable peaks, was accomplished by heating the sample in 80% formamide prior to electrophoresis and the presence of 2-8 M urea in the electrophoresis buffer. Efficient separations were obtained over a wide range of electrical field strengths and temperatures using the gel polymer hydroxypropylmethylcellulose (HPMC) as separation matrix. Low HPMC concentrations (< 0.3%) were suited for the separation of high molecular mass RNA (> 1000 bases) whereas higher HPMC concentrations were required for optimal separation of low molecular mass RNA. An optimized system was applicable for the separation of the predominating RNA populations (small RNA of 60-300 bases (as a group of unseparated peaks), 18S and 28S rRNA) in total RNA from a human glioma cell line. This is the first systematic investigation of electrophoresis of higher molecular mass RNA in capillaries, and motivates further studies to transfer electrophoresis of RNA to the capillary format.

Electrochemistry↗

PubMed1997

High-resolution density gradient electrophoresis of proteins and subcellular organelles.

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.

Electrophoresis, Polyacrylamide Gel↗

PubMed1998

Genotyping of forensic short tandem repeat (STR) systems based on sizing precision in a capillary electrophoresis instrument.

Automated fluorescence analysis of polymerase chain reaction (PCR)-amplified short tandem repeat (STR) systems by capillary electrophoresis (CE) is becoming an established tool both in forensic casework and in the implementation of both state and national convicted offender DNA databases. A new capillary electrophoresis instrument, the ABI Prism 310 Genetic Analyzer, along with the Performance Optimized Polymer 4 (POP-4) provides an automated and precise method for simultaneously analyzing ten fluorescently labeled STR loci from a single PCR amplification kit, which provides a power of discrimination of approximately one in five billion from a single PCR amplification. Data are presented on sizing precision, sizing accuracy, and resolution for the STR loci in the AmpFlSTR Profiler kit. Sizing accuracy is highly dependent on the electrophoresis system, and therefore the reporting of alleles based on the nucleotide size obtained from an electrophoresis system is not recommended for forensic work. The precision of the 310 capillary electrophoresis system, coupled with software developed for automated genotyping of alleles based on the use of an allelic ladder, allows for accurate genotyping of STR loci. Sizing precision of < or = 0.16 nucleotide standard deviation was obtained with this system, thus allowing for accurate genotyping of length variants that differ in length by a single nucleotide.

Alleles↗

PubMed1998

Lectin-gradient gel affinity electrophoresis of glycoproteins.

Lectin-gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4 degrees C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody-affinity blotting was superior for low concentrations of alpha-fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin-gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electrophoresis.

Adult↗

PubMed1998

Nonaqueous capillary electrophoresis--its applicability in the analysis of food, pharmaceuticals and biological fluids.

The use of nonaqueous electrophoresis media for the application of capillary electrophoresis in the analysis of food, pharmaceuticals and biological fluids is reviewed. Some of the applications are discussed in detail and the benefits of using nonaqueous media in these cases are outlined. Three new applications within pharmaceutical analyses are presented. In these methods either a simple sample pretreatment by dilution with methanol (determination of chlorhexidine in a cream) or selective on-line capillary electrophoresis mass spectrometry (methods for identification of seizure drugs or opium alkaloids) are used. The choice of organic solvents and electrolytes for nonaqueous capillary electrophoresis are discussed. Furthermore, validation data obtained using capillary electrophoresis based on the nonaqueous principle are listed and discussed.

Animals↗

PubMed2004

Separation of biomolecules using electrophoresis and nanostructures.

A large number of nanostructures have the potential to be used together with electrophoresis as separation media or separation additive in capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other analytical techniques. Among those structures are nanotubes, nanocavities, nanowires, nanoposts, nanocones, nanospheres, molecular imprints, nanoparachutes (conical monodendrons), and general nanoparticles with random structures. This review is focused only on publications describing experimental works using molecular imprints, nanoposts, and nanospheres that are fabricated and applied for the purpose of separation media in electrophoresis-driven separations. The review follows an approximate chronological order in each section. As shown, the most popular are those resulting from molecular imprinting technologies. These biomimetic receptors are used in a great variety of fields, which includes electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other fields not reviewed in this work. A few examples of these other fields are, e.g., liquid chromatography, membranes, extractor or preconcentration techniques, immunosorbent assays, and sensing devices. The second topic scanned in the present work is the nanostructures that are used as obstacles to replace gels or polymers solutions in electrophoresis. Finally, the nascent field of nanospheres of gold and other materials as separation media is also reviewed.

Electrophoresis, Microchip↗

PubMed2005

The molecular end effect and its critical impact on the behavior of charged-uncharged polymer conjugates during free-solution electrophoresis.

Recently two novel techniques using free-solution electrophoresis to separate charged-uncharged polymer conjugates have proven successful: end-labeled free-solution electrophoresis (ELFSE) for DNA sequencing, and free-solution conjugate electrophoresis (FSCE) for molar mass profiling of uncharged polymers. The approach taken to analyze the experimental data was an extension of the theory of Long and co-workers (Long, D., Dobrynin, A. V., Rubinstein, M., Ajdari, A., J. Chem. Phys. 1998, 108, 1234-1244) for the electrophoresis of molecules with varying charge distributions. This theory also predicts that the ends of the polymers play a large role in determining the polymer's overall mobility; however, this aspect of the theory was neglected in previous work. Until now this "end effect" has, to the knowledge of the authors, not been recognized in experimental data. Through a careful investigation of the predicted end effect and a reanalysis of the experimental data, we demonstrate that indeed this effect critically impacts on the behavior of charged-uncharged polymer conjugates during electrophoresis. This work indicates that not only does the end effect need to be taken into account to avoid significant errors in data analysis, but also it provides novel system optimization approaches.

Electrophoresis↗

PubMed2005

Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples.

The random intergenic spacer analysis is a recently developed technique for the study of microbial populations. The bacterial intergenic spacer (ITS) is located between 16S rRNA and 23S rRNA genes and presents different length and sequence among bacterial species. Therefore, the amplicons can be separated by electrophoresis commonly performed at low voltage during several hours. Although this technique is especially useful for unculturable microorganisms, it has not been applied before to clinical sample analysis. As these samples have a limited number of bacterial species, the size of the gels may be reduced to facilitate their handling and to reduce the running time. To obtain maximum separation among the ITS bands, we analysed in this work different electrophoretical conditions including staircase electrophoresis, a technique based on the application of several voltage steps. The results obtained showed a different behaviour of the electrical resistance during the performance of submarine horizontal and vertical staircase electrophoresis. In the first case the resistance decreased during most of the running time whereas in the second case it increased. Here, we show that the performance of horizontal staircase electrophoresis reduces the running time more than 80% with respect to conventional electrophoresis at low voltages. This procedure was applied to the separation of ITS bands from bacterial DNA present in a tissue from a vocal cord biopsy. The sequencing of these bands allowed their identification. This new procedure may be very useful in the rapid diagnosis of bacteria present in human, animal and plant tissues.

Bacteria↗

PubMed2006

Optimization of capillary array electrophoresis single-strand conformation polymorphism analysis for routine molecular diagnostics.

Mutation screening is widely used for molecular diagnostics of inherited disorders. Furthermore, it is anticipated that the present and future identification of genetic risk factors for complex disorders will increase the need for high-throughput mutation screening technologies. Capillary array electrophoresis (CAE) SSCP analysis is a low-cost, automated method with a high throughput and high reproducibility. Thus, the method fulfills many of the demands to be met for application in routine molecular diagnostics. However, the need for performing the electrophoresis at three temperatures between 18 degrees C and 35 degrees C for achievement of high sensitivity is a disadvantage of the method. Using a panel of 185 mutant samples, we have analyzed the effect of sample purification, sample medium and separation matrix on the sensitivity of CAE-SSCP analysis to optimize the method for molecular diagnostic use. We observed different effects from sample purification and sample medium at different electrophoresis temperatures, probably reflecting the complex interplay between sequence composition, electrophoresis conditions and sensitivity in SSCP analysis. The effect on assay sensitivity from three different polymers was tested using a single electrophoresis temperature of 27 degrees C. The data suggest that a sensitivity of 98-99% can be obtained using a 10% long chain poly-N,N-dimethylacrylamide polymer.

Acrylamides↗

PubMed1993

A nondenaturing preparative gel electrophoresis system for the recovery of functional proteins. Application to the identification of an endogenous protein inhibitor of fucosyl-transferase activities.

Electroelution of protein bands resolved by nondenaturing polyacrylamide gel electrophoresis was performed to identify an endogenous protein inhibitor of fucosyltransferase activities, called fuctinin, through its biological activity. After electrophoresis protein bands were negatively stained with zinc acetate, indicating that this staining technique can be also applied to nondenaturing polyacrylamide gels. However, even under appropriate electroelution conditions, a strong fucosyl-transferase inhibitory activity was eluted from the polyacrylamide gel itself that impeded the measure of fuctinin activity. As an alternative to electroelution, collection of proteins resolved by nondenaturing polyacrylamide gel electrophoresis as they are electrophoresed off the end of the gel was assayed. In the commercially available preparative electrophoretic systems, an elution chamber is limited by a semipermeable membrane on which proteins were adsorbed in the low-ionic-strength buffer used in nondenaturing electrophoresis. We demonstrate that the simple and inexpensive electrofractionation system described by Shain et al. (Anal. Biochem. 200, 47-51, 1992) can be successfully applied under nondenaturing conditions to purify and identify fuctinin through its biological activity. However, caution must be taken in the design of the apparatus in order to avoid local heating and thermal denaturation of proteins. The utility of this nondenaturing preparative electrophoresis system for the study of functional proteins is also demonstrated by the recovery of enzymatic activities of alkaline phosphatase and beta-galactosidase.

Electrophoresis, Polyacrylamide Gel↗

PubMed1996

Determination of intermediates of hemoglobin-haptoglobin complex of haptoglobin polymers by "Crossed hemoglobin electrophoresis".

We designed a "crossed hemoglobin electrophoresis" to be able to detect the intermediates of a hemoglobin-haptoglobin complex without the need for the purification step of haptoglobin molecules. Native polyacrylamide gel electrophoresis was performed as the first dimensional electrophoresis followed by a cross to hemoglobin as the second dimensional electrophoresis. Using this method, we found that a number of intermediates of normal Hp 2-2, Hp 2-1 polymers, particularly those from the trimer to the pentamer, were detected on a polyacrylamide gel. Each of their polymers combined to the hexamer with at least an equimolar content of hemoglobin. In the process of detecting the haptoglobin serotype of more than 10,000 patients for the clinical utilization of quantitative analytical data, we observed two haptoglobin variants, the Hp Johnson and a haptoglobin serotype similar to the Carlberg type. Using our crossed hemoglobin electrophoresis, we also detected the capacity for combining with hemoglobin of each polymer of these variants.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2001

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels.

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.

Animals↗

PubMed1991

Distribution of actin filament lengths and their orientation measured by gel electrophoresis in capillaries.

F-actin was electrophoresed in capillary tubes filled with agarose gel. The use of capillary imparted high resistance on the gel allowing the use of high enough concentration of salts to keep F-actin polymerized, and allowed the application of high electric fields without liberating considerable amount of heat. The intensity profile of the electrophoretic band of F-actin showed a peak, which in 1% agarose in the electric field of 17.8 V cm-1 at 0 degree C, migrated at 3.4 cm hr-1. Microscopic observation of actin filaments extracted from different positions along the gel showed that during electrophoresis filaments distributed themselves in such a manner that the longest polymers migrated slowest and the shortest migrated fastest. Using this observation we calculated the weight and number distributions of filament lengths from corresponding experimental intensity profiles. Phalloidin-labelled F-actin oriented in the gel upon application of an electric field. F-actin showed unusual orientational response: it oriented rapidly when the field was applied, but relaxed very slowly when the field was removed. Orientation of F-actin varied within an electrophoretic band, longest polymers showing the best orientation and short oligomers and monomers not orienting at all. The degree of orientation increased with the size of the electric field. When F-actin was labelled with phalloidin before electrophoresis, it was no longer able to migrate in the gel, but the electric field oriented it in the same way as when it was labelled after the electrophoresis. These results show that the electrophoresis of F-actin in agarose fractionates it according to its length, that by using electrophoresis it is possible to rapidly obtain distribution of filament lengths, and that F-actin migrates in agarose by the process of reptation.

Actins↗

PubMed1983

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture.

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.

Electrophoresis, Polyacrylamide Gel↗

PubMed1987

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks.

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.

DNA↗