Electrophoretic migration of proteins in semidilute polymer solutions

We present a systematic study of the electrophoretic migration of 10-200 kDa protein fragments in dilute-polymer solutions using microfluidic chips. The electrophoretic mobility and dispersion of protein samples were measured in a series of monodisperse polydimethylacrylamide (PDMA) polymers of different molecular masses (243, 443, and 764 kDa, polydispersivity index <2) of varying concentration. The polymer solutions were characterized using rheometry. Prior to loading onto the microchip, the polymer solution was mixed with known concentrations of SDS (SDS) surfactant and a staining dye. SDS-denatured protein samples were electrokinetically injected, separated, and detected in the microchip using electric fields ranging from 100 to 300 V/cm. Our results show that the electrophoretic mobility of protein fragments decreases exponentially with the concentration c of the polymer solution. The mobility was found to decrease logarithmically with the molecular weight of the protein fragment. In addition, the mobility was found to be independent of the electric field in the separation channel. The dispersion is relatively independent of polymer concentration and it first increases with protein size and then decreases with a maximum at about 45 kDa. The resolution power of the device decreases with concentration of the PDMA solution but it is always better than 10% of the protein size. The protein migration does not seem to correspond to the Ogston or the reptation models. A semiempirical expression for mobility given by van Winkle fits the data very well.     

Effect of temperature on the separation of long DNA fragments in polymer solution

Electrophoresis of long DNA fragments in polymer solutions is still attractive when performed in short capillaries. Then the separations can be accomplished in minutes rather than hours as is usual in various slab electrophoresis techniques. In this paper we focused on the behavior of large DNA fragments in pulsed field capillary electrophoresis under various temperature conditions. The mobility dependence of fragments of lambda-DNA single-cut mixture on various frequencies at three different temperatures showed that the antiresonance mobility minima are shifted to higher frequencies at higher temperatures. This interesting result is explained in terms of the geometration model of DNA motion.     

Anomalously fast migration of triplet-repeat DNA in capillary electrophoresis with linear polymer solution

We investigated the capillary electrophoretic behavior of single-stranded DNA fragments in methylcellulose solution, and found that triplet-repeat DNA showed anomalously faster mobilities than DNA markers with random sequence. Through the further study on the electrophoretic data, reptation model is proven appropriate to describe the migration of DNA under our experimental conditions. Accordingly, with the equations based on reptation theory, we could obtain the persistence length of DNA fragments and find that these values of triplet-repeat DNAs are larger than that of DNA markers with random sequence, which means the former DNAs are less flexible than the latter ones when they migrate in the electric field. This phenomenon is supposed to result from the characteristic higher-order structure formed by GC base pairs within triplet-repeat DNA, which is further proven by the resumed migration order in accordance with DNA size when the denaturant is added into the sieving matrix.     

Electrophoretic dynamics of large DNA stars in polymer solutions and gels

We have developed a procedure for synthesizing large stable branched DNA structures that enables visualization via fluorescence microscopy. Using this procedure we have synthesized large DNA stars and observed their electrophoretic behavior in polymer solutions and gels. In dilute polyacrylamide solutions, the DNA stars move as random coils and appear to experience only brief collisions with the polymer chains in solution. The effect of polymer solution concentration on the electrophoretic mobility of stars in the dilute regime is found to be in good accord with predictions of the transient entanglement coupling (TEC) model. In semidilute polymer solutions, the star arms extend in the field direction and drag the core through the matrix. The star arms form several U-shaped conformations as they collide and engage with polyacrylamide chains. The U-shaped conformations occasionally evolve into J-shaped conformations as the star arms slide off the matrix chains they engage during electrophoretic migration. In concentrated polymer solutions, the arms of the star extend and form V-shaped structures with the core as the apex. The arms then pull the core through the matrix. These V-shaped conformations are much longer-lived than U-shaped ones and, unlike the latter, do not transform to J-shaped conformations. In polyacrylamide and agarose gels, where matrix entanglements are fixed, DNA stars become trapped when entanglements with matrix molecules prevent the core from being pulled through the matrix by the extended arms. This trapping was observed at all gel concentrations and electric fields studied.     

Electrophoretic mobility of linear and star-branched DNA in semidilute polymer solutions

Electrophoresis of large linear T2 (162 kbp) and 3-arm star-branched (N(Arm) = 48.5 kbp) DNA in linear polyacrylamide (LPA) solutions above the overlap concentration c* has been investigated using a fluorescence visualization technique that allows both the conformation and mobility mu of the DNA to be determined. LPA solutions of moderate polydispersity index (PI approximately 1.7-2.1) and variable polymer molecular weight Mw (0.59-2.05 MDa) are used as the sieving media. In unentangled semidilute solutions (c* < c < c(e)), we find that the conformational dynamics of linear and star-branched DNA in electric fields are strikingly different; the former migrating in predominantly U- or I-shaped conformations, depending on electric field strength E, and the latter migrating in a squid-like profile with the star-arms outstretched in the direction opposite to E and dragging the branch point through the sieving medium. Despite these visual differences, mu for linear and star-branched DNA of comparable size are found to be nearly identical in semidilute, unentangled LPA solutions. For LPA concentrations above the entanglement threshold (c > c(e)), the conformation of migrating linear and star-shaped DNA manifest only subtle changes from their unentangled solution features, but mu for the stars decreases strongly with increasing LPA concentration and molecular weight, while mu for linear DNA becomes nearly independent of c and Mw. These findings are discussed in the context of current theories for electrophoresis of large polyelectrolytes.     

Interaction of DNA fragments with counterions in aqueous solution

Monte Carlo simulation of the hydration of metal ion—DMP⁻ and metal ion—9-methylguanine complexes was performed. A comparative analysis of the results for Na⁺ and K⁺ ions was carried out. The main stages of dissociation were revealed. The energy effects of dissociation were evaluated. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 11, pp. 2174–2177, November, 1998.     

Electrophoretic behavior of plasmid DNA in the presence of various intercalating dyes

In the present study, the electrophoretic behavior of linear, supercoiled and nicked circular plasmid DNA in the presence of various intercalating dyes was characterized using pGL3 plasmid DNA as a model. The enzymatic digestion of pGL3 plasmid DNA with HindIIIwas monitored by capillary electrophoresis coupled with laser-induced fluorescence detection (CE-LIF). Nicked circular plasmid DNA was found to be relatively sensitive to enzymes, and was almost digested into the linear conformer after 10-min incubation, indicating that nicked circular plasmid DNA has little chance of targeting and entering the cell nucleus. Partly digested plasmid DNA containing only linear and supercoiled conformers can be used as a standard to confirm the migration order of plasmid DNA. In methylcellulose (MC) solution with YO-PRO-1 or YOYO-1, linear plasmid DNA eluted first, followed by supercoiled and nicked plasmid DNA, and nicked plasmid DNA eluted as a broad peak. With SYBR Green 1, nicked plasmid DNA eluted first as three sharp peaks, followed by linear and supercoiled plasmid DNA. The nuclear plasmid DNA from two transfected cell lines was successfully analyzed using the present procedure. Similar results were obtained with an analysis time of seconds using microchip electrophoresis with laser-induced fluorescence detection (mu-CE-LIF). To our knowledge, these results represent the first reported analysis of nuclear plasmid DNA from transfection cells by CE-LIF or mu-CE-LIF without pre-preparation, suggesting that the present procedure is a promising alternative method for evaluating transfection efficiency of DNA delivery systems.     

一种咪唑啉结构疏水缔合聚合物合成与溶液性质

通过自由基胶束共聚法,在氧化还原引发体系下以丙烯酰胺(AM),丙烯酸钠(Na AA),二甲基二烯丙基氯化铵(DMDAAC)及1-(2-N-烯丙基氨乙基)-2-油酸基咪唑啉(NIPA)共聚合成了一种含咪唑啉结构的缔合聚合物。考察了单体配比、p H及引发剂浓度等条件对聚合反应的影响。通过FT-IR、1H NMR分析对聚合物进行了结构表征。实验发现:该聚合物具有较好的耐温性、抗剪切及抗盐性能(120oC:其粘度保留率达28.12%;1000 s-1:其粘度保留率达16.74%;16000 mg/L的Na Cl:粘度保留率为25.99%;2600 mg/L1的Mg Cl2:粘度保留率为22.48%;2600 mg/L的Ca Cl2:粘度保留率为18.10%)。在室内模拟岩芯驱替实验中,该聚合物可提高采收率16.65%。     

The migration anomaly of DNA fragments in polyacrylamide gels allows the detection of small sequence-specific DNA structure variations

Curved DNA fragments have a reduced electrophoretic mobility in polyacrylamide gels. The retardation in gels is extremely sensitive to small structural variations which influence the DNA helix axis. This gel assay can also be used to detect very small structural variations in DNA sequences which are not curved: The noncurved sequences of interest can be combined with curved stretches in phase with the helix turn. Using such sequence constructions, even subtle influences on the DNA helix axis can be detected. Experiments of this kind allow the determination of a relative order of sequence-specific DNA twist and wedge angles.     

Mannitol influence on the separation of DNA fragments by capillary electrophoresis in entangled polymer solutions

A new kind of sieving matrix is presented in this paper to allow satisfactory separation of DNA fragments in a relatively low viscous solution. When a certain amount of mannitol was added to cellulose solution not concentrated enough to separate PGEM-3Zf(+)/HaeIII standards well, a polymer solution with low viscosity but with very good separation effects was obtained. The separation result of this sieving buffer was comparable with those using highly concentrated cellulose solutions. The sieving ability of solutions with different cellulose concentrations and different amounts of mannitol has been investigated. It was proved that 0.5% was the minimum hydroxypropylmethylcellulose (HPMC) concentration that could be used to separate DNA fragments satisfactorily. HPMC solutions with a concentration of less than 0.5% could not separate the standard DNA fragments even in the presence of mannitol. It was found that 6% was the optimized mannitol concentration because either more or less mannitol will lead a decrease of resolution. The principle of the positive influence of mannitol has also been discussed.     

Divergent dispersion behavior of ssDNA fragments during microchip electrophoresis in pDMA and LPA entangled polymer networks

Resolution of DNA fragments separated by electrophoresis in polymer solutions ("matrices") is determined by both the spacing between peaks and the width of the peaks. Prior research on the development of high-performance separation matrices has been focused primarily on optimizing DNA mobility and matrix selectivity, and gave less attention to peak broadening. Quantitative data are rare for peak broadening in systems in which high electric field strengths are used (>150 V/cm), which is surprising since capillary and microchip-based systems commonly run at these field strengths. Here, we report results for a study of band broadening behavior for ssDNA fragments on a glass microfluidic chip, for electric field strengths up to 320 V/cm. We compare dispersion coefficients obtained in a poly(N,N-dimethylacrylamide) (pDMA) separation matrix that was developed for chip-based DNA sequencing with a commercially available linear polyacrylamide (LPA) matrix commonly used in capillaries. Much larger DNA dispersion coefficients were measured in the LPA matrix as compared to the pDMA matrix, and the dependence of dispersion coefficient on DNA size and electric field strength were found to differ quite starkly in the two matrices. These observations lead us to propose that DNA migration mechanisms differ substantially in our custom pDMA matrix compared to the commercially available LPA matrix. We discuss the implications of these results in terms of developing optimal matrices for specific separation (microchip or capillary) platforms.     

Electrophoretic behavior of cerebellar granule neurons

The electrophoresis of cerebellar granule neurons is observed, and a theoretical model proposed to simulate its electrophoretic behavior. We assume that the surface of a neuronal cell carries dissociable acidic functional groups, and the liquid phase contains a mixed (a:b) + (c:b) electrolyte, where a and c are the valences of cations and b is the valence of anions. The cations of valence c are allowed to bind to dissociated functional groups. The model proposed is readily applicable to the prediction of the surface properties of cerebellar granule neurons such as the density of dissociable functional groups and the equilibrium constant of the dissociation reaction. The applicability of the present model is justified by fitting it to the measured electrophoretic mobility data.     

Electrophoretic capture of circular DNA in gels

Results on electrophoretic capture of circular DNA in porous gels are reviewed. Processes which cause arrest of circular forms of DNA during electrophoresis can provide very efficient separation mechanism for the purification of plasmids and bacterial artificial chromosomes if the corresponding linear form is not trapped and therefore removed by the electric field. Two types of such topological traps have been proposed, impalement and lobster traps, and we here review the present experimental support for the existence of these two circle-specific mechanisms. Experiments designed to characterize the traps are discussed, regarding the concentration of the traps as well as their efficiency and capacity to trap both relaxed and supercoiled circular DNA. Studies of the dynamics of the capture process show that the average capture time is on the order of 10 s at 20 V/cm, by which time the circles have migrated several hundred micrometers and have passed hundreds of traps. We also review results on attempts to improve the capacity and efficiency of the trapping process by modification of the gels either by enzymatic treatment or by cogelation of neutral polymers.     

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.     

Electrophoretic analysis and purification of fluorescent single-walled carbon nanotube fragments

Arc-synthesized single-walled carbon nanotubes have been purified through preparative electrophoresis in agarose gel and glass bead matrixes. Two major impurities were isolated: fluorescent carbon and short tubular carbon. Analysis of these two classes of impurities was done. The methods described may be readily extended to the separation of other water-soluble nanoparticles. The separated fluorescent carbon and short tubule carbon species promise to be interesting nanomaterials in their own right.     

Electrophoretic NMR studies of polymer and surfactant systems

The aim of this tutorial review is to introduce to a broader readership the emerging technique of electrophoretic NMR (eNMR). The "electrophoretic" element of the technique refers to the fact that charged particles are induced to flow by the application of an electric field. This flow is measured using pulsed-gradient spin-echo NMR (PGSE-NMR). The great potential of this experimental approach is the fact that NMR is chemically selective and non-invasive. eNMR, especially when combined with the more established PGSE-NMR experiment, may therefore be used to quantify the structure of multi-component systems via the dynamics and charge of each species within a complex mixture. Accordingly, eNMR is likely to be of great significance for colloid scientists, biologists, technologists and formulation scientists.     

Electrophoretic properties of dodecyltrimethylammonium bromide micelles in KBr solution

Charge in ionic micelles determines the trends of their stability and their practical applications. Charge can be calculated from zeta potential (zeta) measurements, which, in turn, can be obtained by Doppler microelectrophoresis. In this study, the electrophoretic properties of dodecyltrimethylammonium bromide (DTAB) in KBr aqueous solution (0-6 mM) were determined by Doppler microelectrophoresis. At very low surfactant concentrations (up to 6 mM), zeta potential was quite constant and due to the ionized monomers (DTA+). Above 6 mM, zeta potential increased to a maximum at surfactant concentrations still below the critical micellar concentration (CMC). This increase could be explained by a formation of nonmicellar aggregates of DTAB. Then, above the CMC, zeta potential underwent an abrupt reduction, which was dependent qualitatively and quantitatively on KBr concentration, and which could be due to an increase of the number of counterions adsorbed on the micelle surface. Calculation of effective micellar charge from zeta potential gave the surface charge density. Comparing this value with the theoretical, obtained from geometrical considerations, a fraction of 0.29 of charged micellar headgroups was obtained when DTAB was in aqueous solution, which is consistent with the value obtained by conductivity measurements.     

Densitometric DNA Analysis in Agarose Electrophoretic Gels

JPC – Journal of Planar Chromatography – Modern TLC - Agarose gel electrophoresis is a basic separation tool used in molecular biology, mostly for qualitative DNA analysis. There are...