Computer-assisted 2-D agarose electrophoresis of Haemophilus influenzae type B meningitis vaccines and analysis of polydisperse particle populations in the size range of viruses: a review

When protein-polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices.     

Electrophoretic size separations in liquified agarose of polystyrene particles and circular DNA.

Polystyrene sulfate particles of 0.37 to 1.78 mu in diameter are retarded in their electrophoretic migration in proportion to the concentration of agarose liquified above its gelling temperature. In the concentration range of 0.02 to 0.2% liquified agarose, the degree of this retardation in electrophoresis at 40 degrees C is inversely related to particle size. By contrast, mitochondrial DNA (16 kb), plasmid pBR322 DNA (4 kb) and plasmid PSA509 DNA (3 kb) exhibit under the same conditions a degree of retardation which is proportional to their size. This confirms the existence of two divergent mechanisms of size separation similarly observed in other liquid polymer media, i.e. one based on collisions with the gel fiber (molecular sieving) and one based on exclusion from the fiber network (the electrophoretic equivalent of gel permeation).     

Electrophoretic properties of BSA-coated quantum dots

Low toxic InP/ZnS quantum dots (QDs), ZnS:Mn²⁺/ZnS nanocrystals and CdSe/ZnS nanoparticles were rendered water-dispersible by different ligand-exchange methods. Eventually, they were coated with bovine serum albumin (BSA) as a model protein. All particles were characterised by isotachophoresis (ITP), laser Doppler velocimetry (LDV) and agarose gel electrophoresis. It was found that the electrophoretic mobility and colloidal stability of ZnS:Mn²⁺/ZnS and CdSe/ZnS nanoparticles, which bore short-chain surface ligands, was primarily governed by charges on the nanoparticles, whereas InP/ZnS nanocrystals were not charged per se. BSA-coated nanoparticles showed lower electrophoretic mobility, which was attributed to their larger size and smaller overall charge. However, these particles were colloidally stable. This stability was probably caused by steric stabilisation of the BSA coating.     

Ultra-thin-layer agarose gel electrophoresis. I. Effect of the gel concentration and temperature on the separation of DNA fragments

A novel, rapid and efficient separation method is described for the analysis of double stranded (ds) DNA fragments in the form of horizontal ultra-thin-layer agarose gel electrophoresis. This separation technique combines the multilane, high-throughput separation format of agarose slab gel electrophoresis with the excellent performance of capillary electrophoresis. The electrophoretic separation of the fluorophore (Cy5)-labeled dsDNA molecules were imaged in real time by a scanning laser-induced fluorescence/avalanche photodiode detection system. Effects of the gel concentration (Ferguson plot) and separation temperature (Arrhenius plot) on the migration characteristics of the DNA fragments are discussed. An important genotyping application is also shown by characterizing the polymorphic region (2× or 4×48 base pair repeats) of the dopamine D₄ receptor gene (D₄DR, exon III region) for ten individuals, using PCR technology with Cy5-labeled primers and ultra-thin-layer agarose gel electrophoresis.     

Capillary electrophoresis in agarose solutions: extension of size separations to DNA of 12 kb in length.

The upper limit of the size range of DNA amenable to separation in agarose solutions above their gelling temperature, using capillary zone electrophoresis apparatus, was increased to 12 kb. The plot of log(bp) vs. mobility derived from electrophoresis in 1.7% agarose solution is biphasic, exhibiting higher resolving power for DNA less than 1 kb in size than that of larger sizes. Resolving power for DNA larger than 1 kb increased when the agarose concentration was increased in the range of 1.0-2.6%. It was similar in solutions at 40 degrees C of SeaPrep and SeaPlaque agaroses as well as in Acrylaide (trade names are those of the manufacturer). However, the resolving power of SeaPrep agarose at 25 degrees C was inferior to that at 40 degrees C. Concave plots of log(mobility) vs. concentration of the agarose solutions are those predicted under the assumption that the effective "equivalent radius" of the DNA molecule diminishes with increasing agarose concentration in the investigated concentration range up to 2.6%.     

The electric field dependence of DNA mobilities in agarose gels: a reinvestigation

The electric field dependence of the electrophoretic mobility of linear DNA fragments in agarose gels was reinvestigated in order to correct the observed mobilities for the different temperatures actually present in the gel during electrophoresis in different electric field gradients. When corrected to a common temperature, the electrophoretic mobilities of DNA fragments less than or equal to 1 kilobase pairs (kbp) in size were independent of electric field strength at all field strengths from 0.6 to 4.6 V/cm if the gels contained less than or equal to 1.4% agarose. The mobilities of larger DNA fragments increased approximately linearly with electric field strength. If the agarose concentration was higher than 2%, the mobilities of all DNA fragments increased with increasing electric field strength. The electric field dependence of the mobility was larger in gels cast and run in Tris-borate buffer (TBE) than in gels cast and run in Tris-acetate buffer (TAE), and was more pronounced in gels without ethidium bromide incorporated in the matrix. Ferguson plots were constructed for the various DNA fragments, both with and without extrapolating the temperature-corrected mobilities to zero electric field strength. Linear Ferguson plots were obtained for all fragments less than or equal to 12 kbp in size in agarose gels less than or equal to 1.4% in concentration if the mobilities were first extrapolated to zero electric field strength. Concave upward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 2 kbp in size at finite electric field strengths. Convex downward curvature of the Ferguson plots was observed for DNA fragments greater than or equal to 1 kbp in size in agarose gels greater than or equal to 2% in concentration. The mobilities of the various DNA fragments, extrapolated to zero agarose concentration and zero electric field strength, decreased with increasing DNA molecular weight; extrapolating to zero molecular weight gave an "intrinsic" DNA mobility of 2.7 x 10(-4) cm2/Vs at 20 degrees C. The pore sizes of LE agarose gels cast and run in TAE and TBE buffers were estimated from the mobility of the DNA fragments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

Ultrathin-layer sodium dodecyl sulfate gel electrophoresis of proteins: effects of gel composition and temperature on the separation of sodium dodecyl sulfate-protein complexes

This paper discusses the effects of gel composition and separation temperature on the migration properties of fluorescein-5-isothiocyanate-labeled protein molecular mass markers (ranging from 20 100 to 205 000 Da) in automated ultrathin-layer sodium dodecyl sulfate (SDS) gel electrophoresis. The separation mechanism with the agarose and composite agarose - linear polyacrylamide, agarose - hydroxyethyl cellulose, and agarose - polyethylene oxide matrices were all found to comply with the Ogston sieving model in the molecular mass range of the protein molecules investigated. Our temperature studies revealed that electrophoretic separation of SDS protein complexes is an activated process and, in pure agarose and in composite agarose hydroxyethyl cellulose and agarose - polyethylene oxide matrices that the separation requires increasing activation energy as a function of the molecular mass of the separated proteins. On the other hand, when linear polyacrylamide was used as composite additive, the activation energy demand of the separation decreased with increasing solute molecular mass. The sensitivity of the laser-induced fluorescent detection of the automated ultrathin-layer electrophoresis system was evaluated by injecting a series of dilutions of the markers and was found to be less than 2.5 ng/band for the fluorophore-labeled protein.     

Nonlinear electrophoresis of dielectric particles in Newtonian fluids

In classical electrokinetics, the electrophoretic velocity of a dielectric particle is a linear function of the applied electric field. Theoretical studies have predicted the onset of nonlinear electrophoresis at high electric fields because of the nonuniform surface conduction over the curved particle. However, experimental studies have been left behind and are insufficient for a fundamental understanding of the parametric effects on nonlinear electrophoresis. We present in this work a systematic experimental study of the effects of buffer concentration, particle size, and particle zeta potential on the electrophoretic velocity of polystyrene particles in a straight rectangular microchannel for electric fields of up to 3 kV/cm. The measured nonlinear electrophoretic particle velocity is found to exhibit a 2(±0.5)-order dependence on the applied electric field, which appears to be within the theoretically predicted 3- and 3/2-order dependences for low and high electric fields, respectively. Moreover, the obtained nonlinear electrophoretic particle mobility increases with decreasing buffer concentration (for the same particle) and particle size (for particles with similar zeta potentials) or increasing particle zeta potential (for particles with similar sizes). These observations are all consistent with the theoretical predictions for high electric fields.     

Highly efficient approach for characterizing nanometer-sized gold particles by capillary electrophoresis

This paper demonstrates that capillary electrophoresis (CE) can be employed for characterizing the sizes of nanometer-scale gold particles. We characterized the gold nanoparticles by effecting CE separation using a buffer of SDS (70 mM) and 3-cyclohexylamino-1-propanesulfonic acid (CAPS; 10 mM) at pH 11.0 and an applied voltage of 18 kV and obtained a linear relationship (R² > 0.99) between electrophoretic mobilities and size for nanoparticles whose diameters fall in the regime from 5.0 ± 0.5 to 41.2 ± 3.3 nm; the relative standard deviations of these electrophoretic mobilities are <0.8%. We evaluated the feasibility of employing these separation conditions for the size characterization by of gold nanoparticle samples that were synthesized by a rapid microwave heating method. We confirmed that this CE method is a valid one for size characterization by comparing the results obtained by CE with those provided by scanning electron microscopy (SEM); a good correlation exists between these two techniques. Our results demonstrate that CE can be employed to accelerate the analysis of the sizes of nanomaterials.     

Determination of Triton X-100 in influenza vaccine by high-performance liquid chromatography and capillary electrophoresis

Triton X-100 is applied to influenza vaccines at different stages of the manufacturing process to prevent aggregation and precipitation of biomolecules. Furthermore it is used to disintegrate the virus particles in split vaccine and to guarantee the homogeneity during production and utilisation. The final concentration of Triton X-100 has to be determined because the concentration changes in manufacturing process. The determination of the total amount of Triton X-100 as well as the separation of its ethylene oxide oligomers was possible with high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). In HPLC a change of the column and eluent was necessary, in CE different electrolytes were used for the various separation effects. The HPLC method for the analysis of total Triton was preferred for the quantification of Triton X-100 in influenza vaccine because of better linearity, reproducibility and detection sensitivity compared to CE. In the end products an average concentration of 0.117 mg/mL was found. Received: 19 December 1996 / Revised: 27 February 1997 / Accepted: 6 March 1997     

Electrophoretic size separation of particles in a periodically constricted microchannel

The size separation of Brownian particles with the same free mobility in an electrophoretic microchannel with alternating thick regions and narrow constrictions is studied theoretically. The electrophoretic mobility is field dependent and generally increases with field strength. In weak fields, Brownian diffusion dominates and the migration is controlled by the entrance effect. Therefore, smaller particles migrate faster than larger ones. In strong fields, however, the particle tends to follow electric field lines. Smaller particles are susceptible to Brownian motion and thus influenced by the nonuniform electric field in the well significantly. As a result, larger particles possess higher mobilities. Our simulation results agree with the experimental observations and provide guidance for efficient nanofluidic separation.     

电泳技术在纳米颗粒分离中的应用

合成纳米颗粒常在尺寸和形状方面具有广泛分布.在很多实验中,需要利用一定大小及形状的纳米颗粒的独特物理化学性质,因此,简便快速的纳米颗粒分离技术越来越受到诸多科学领域的重视.电泳技术以其高分辨率,被广泛用于多种生物大分子如核酸、蛋白质等的分离纯化.纳米颗粒在尺寸上与生物体中的蛋白复合物、细胞器和微生物等十分接近,考虑到带电纳米颗粒与生物分子在电场中的运动行为的相似性,运用电泳技术进行纳米颗粒的鉴定、分离和纯化是一种新的思路,并取得了良好的实验结果.本文主要介绍了琼脂糖凝胶电泳、毛细管电泳以及其他一些电泳技术在纳米颗粒分离中的研究进展.     

Bicontinuous cubic phase of monoolein and water as medium for electrophoresis of both membrane-bound probes and DNA

Porous hydrogels such as agarose are commonly used to analyze DNA and water-soluble proteins by electrophoresis. However, the hydrophilic environment of these gels is not suitable for separation of important amphiphilic molecules such as native membrane proteins. We show that an amphiphilic liquid crystal of the lipid monoolein and water can be used as a medium for electrophoresis of amphiphilic molecules. In fact, both membrane-bound fluorescent probes and water-soluble oligonucleotides can migrate through the same bicontinuous cubic crystal because both the lipid membrane and the aqueous phase are continuous. Both types of analytes exhibit a field-independent electrophoretic mobility, which suggests that the lipid crystal structure is not perturbed by their migration. Diffusion studies with four membrane probes indicate that membrane-bound analytes experience a friction in the cubic phase that increases with increasing size of the hydrophilic headgroup, while the size of the membrane-anchoring part has comparatively small effect on the retardation.     

The distribution of particles characterized by size and free mobility within polydisperse populations of protein-polysaccharide conjugates, determined from two-dimensional agarose electropherograms

New approaches for the characterization of polydisperse particle populations are presented*. The investigated samples contain virus-sized protein-polysaccharide conjugates which had previously been prepared as immunogens against bacterial meningitis (Hib). The analysis is based on two-dimensional agarose electrophoresis (Serwer-type). This method, like the one of O'Farrell, achieves a separation according to size and charge. It relies on a different principle, however, and is applicable to nondenatured particles which are 100 to more than 1000 times larger in mass than regular uncrosslinked proteins. Data from stained gel patterns are evaluated by the computer program ELPHOFIT, which makes it possible to standardize the gel and to construct a nomogram which defines every position on the gel in terms of particle size and free mobility (related to surface net charge density). The output of ELPHOFIT, consisting of nomogram parameters, is transferred to the image processing program GELFIT. This software is used to evaluate the computer images obtained by digitizing the stained gel patterns: (i) The nomogram is electronically superimposed on the computer image. (ii) The gel pattern is transformed from a curvilinear to a rectangular coordinate system of particle size and free mobility. The center of gravity as well as density maxima are given in coordinates of particle size and free mobility. Ranges of grey levels can be accentuated by adding 16 pseudocolors. (iii) Using surface-stripping techniques, GELFIT provides an estimate for the number of major subpopulations within each preparation. (iv) Numerical values for the distribution of particle size and free mobility are determined. Using program IMAGE, the quantitative physical assessment of a given conjugate preparation is presented in the form of a computer-generated three-dimensional plot, the shape of which serves to identify and characterize the preparation visually. The data analysis based on digitized two-dimensional gel patterns is automated to an extent that a technician can perform routine evaluations. It uses the Macintosh II personal computer.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Effect of the matrix on DNA electrophoretic mobility

DNA electrophoretic mobilities are highly dependent on the nature of the matrix in which the separation takes place. This review describes the effect of the matrix on DNA separations in agarose gels, polyacrylamide gels and solutions containing entangled linear polymers, correlating the electrophoretic mobilities with information obtained from other types of studies. DNA mobilities in various sieving media are determined by the interplay of three factors: the relative size of the DNA molecule with respect to the effective pore size of the matrix, the effect of the electric field on the matrix, and specific interactions of DNA with the matrix during electrophoresis.     

Superfast electrophoresis of electron-type conducting particles

The features of concentration polarization caused by electric current through a unipolar conductive particle are considered. The peculiarities of the formation of an induced space charge near a particle with electron-type conductivity are analysed. It has been shown that the theoretical values of electrophoretic velocity for these particles are essentially smaller than those calculated for particles with ion-type conductivity.A new method to observe the superfast electrophoresis is developed. The electrophoretic velocity of graphite and activated carbon particles of different size (diameter, 200–500 μm) displaced in distilled water and electrolyte solutions in strong electric fields (100–500 V cm⁻¹) was measured. It is shown that, in contrast to classical electrophoresis, the electrophoretic mobility of such particles increases with the particle size and the external field strength. The experimental and theoretical results are compared. The discrepancy between theory and experiment is analysed.     

A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis

We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.     

Isolation of Xis Gen Fragment of λ Phage from Agarose Gel Using Magnetic Particles for Subsequent Enzymatic DNA Sequencing

Gel electrophoresis is one of the most important methods used in biochemistry and molecular biology. The recovery of analytes from the gel required for subsequent analysis including amplification by polymerase chain reaction (PCR) or DNA sequencing is an issue due to the gel contamination. Among the other methods used for sample recovery, the application of nanomaterials is also being investigated. In this study, the applicability of magnetic particles (1 μm) for isolation of DNA fragment from agarose gel with subsequent DNA sequencing was investigated. Electrochemical analysis and DNA sequencing was used to investigate the recovery yield. The influence of dilution of the gel prior to the purification was investigated and the linear dependence with regression coefficient R ² = 0.9972 was obtained using square wave voltammetry. Moreover, bioinformatic analysis was used for comparison of obtained sequences, and simple and easy identification of non-systematic errors caused by both fluorescence labeling reaction and electrophoretic separation. It was found that magnetic nanoparticles based isolation markedly lowered the errors occurring during sequencing of the isolated DNA fragment from 7 to 1 %.