Constant-field Electrophoresis Research Articles

Increase in local protein concentration by field-inversion gel electrophoresis

BackgroundProteins that migrate through cross-linked polyacrylamide gels (PAGs) under the influence of a constant electric field experience negative factors, such as diffusion and non-specific trapping in the gel matrix. These negative factors reduce protein concentrations within a defined gel volume with increasing migration distance and, therefore, decrease protein separation efficiency. Enhancement of protein separation efficiency was investigated by implementing pulsed field-inversion gel electrophoresis (FIGE).ResultsSeparation of model protein species and large protein complexes was compared between FIGE and constant field electrophoresis (CFE) in different percentages of PAGs. Band intensities of proteins in FIGE with appropriate ratios of forward and backward pulse times were superior to CFE despite longer running times. These results revealed an increase in band intensity per defined gel volume. A biphasic protein relative mobility shift was observed in percentages of PAGs up to 14%. However, the effect of FIGE on protein separation was stochastic at higher PAG percentage. Rat liver lysates subjected to FIGE in the second-dimension separation of two-dimensional polyarcylamide gel electrophoresis (2D PAGE) showed a 20% increase in the number of discernible spots compared with CFE. Nine common spots from both FIGE and CFE were selected for peptide sequencing by mass spectrometry (MS), which revealed higher final ion scores of all nine protein spots from FIGE. Native protein complexes ranging from 800 kDa to larger than 2000 kDa became apparent using FIGE compared with CFE.ConclusionThe present investigation suggests that FIGE under appropriate conditions improves protein separation efficiency during PAGE as a result of increased local protein concentration. FIGE can be implemented with minimal additional instrumentation in any laboratory setting. Despite the tradeoff of longer running times, FIGE can be a powerful protein separation tool.

Adaptive response of a new radioresistant strain of Chlamydomonas reinhardtii and correlation with increased DNA double-strandbreak rejoining

In order to study the relationship between radioresistance and the adaptive response, we aimed to produce a new strain of Chlamydomonas reinhardtii with characteristics of high radioresistance coupled with a protoplast structure typical for the genus, and the cell-wall-less phenotype to facilitate rapid cell lysis in DNA double-strand break (DSB) assays. The adaptive response of the new strain was investigated using clonogenic and DSB assays. Strain H-3 was derived by mating a radioresistant strain (AK-9-9) with the cell-wall-less mutant CW15 strain and selecting for radioresistance by clonogenic assay. The random amplification of polymorphic DNA (RAPD) molecular marker system was used to evaluate genetic polymorphisms between H-3 and other related C. reinhardtii strains. DSB were estimated using constant-field electrophoresis. Of several mutant strains tested, strain H-3 was shown to be most radioresistant on the basis of dose to give a 90% lethality (LD90) rate and dose to give a 99% lethality rate (LD99). In addition to its high radioresistance and thinner cell wall as compared with that of the other parental strain AK-9-9, H-3 also expressed a radiation-induced adaptive response measured by clonal survival when given a priming dose before a test dose. DSB were also rejoined more rapidly in cells exposed to a priming dose 4 h previously. It is concluded from split-dose experiments that the already highly radioresistant strain H-3 is further capable of ‘over recovery’ or adaptation to radiation exposure. Accelerated DSB rejoining in cells given a priming dose may underlie the cellular adaptive response in this organism.

Cyclic capillary electrophoresis.

A strategy is described here for increasing both the resolution and the flexibility of capillary electrophoresis performed in a sieving medium of ungelled polymer. This strategy is based on analysis and, sometimes, re-analysis that is done in several stages of constant-field electrophoresis. Enhancement-stages are between the analysis-stages. An enhancement-stage (i) increases the separation between peaks, while (ii) moving DNA molecules in the reverse direction. An enhancement-stage is based on an electrophoretic ratchet generated by a pulsed electrical field that can be zero-integrated. The ratchet-generating pulses are longer than the field pulses that have previously been used to improve the resolution of DNA molecules. No limit has been found to the resolution enhancement achievable. Apparently, diffusion-induced peak broadening is inhibited and, in some cases, may be reversed by the ratchet. The enhancement-stages are critically dependent on the electrical field-dependence of a plot of electrophoretic mobility as a function of DNA length. To generate the pulsed electrical field, a computer-controlled system with a time resolution of 30 microseconds has been developed. Programming is flexible enough to embed other pulses within ratchet-generating pulses. These other pulses can be either the previously used, shorter field-inversion pulses or high-frequency periodic oscillations previously found to sharpen peaks.

The effect of serum albumin on the radiolysis of DNA studied by constant field electrophoresis and compared to alterations caused by low molecular weight OH scavengers

After radiolysis of calf thymus DNA in 10 2 mo ldm 3 phosphate buffer at pH 7 under N2, N2 O and air the yields of double-strand breaks (dsb) have been determined by constant field electrophoresis. Double-strand (dsb) breaks were formed according to a linear-quadratic relationship with dose showing a lower G-value under aerobic than under anaerobic conditions (G (air) 1.4 nmolJ1; G (N2) 2.1 nmol J 1; G (N2O) 4.9 nmolJ1). To test the reliability of this system the effect of low molecular weight OH scavengers which were already used in comparable work with plasmid DNA were studied. The results with plasmid DNA and calf thymus DNA obtained by different techniques of electrophoresis agreed quite well. Under N2 more protection was obtained with ethanol than with DMSO or with t-butanol. Under air, double-strand breakage was further decreased and reached the same level with all of these scavengers. Furthermore the constant field electrophoresis gives similar results as the low-angle light scattering technique for radiation induced double strand breakage of calf thymus DNA. When BSA was used at the same scavenger capacity as the low molecular weight scavengers, the protection against double strand breakage was less if radiolysis was carried out in the presence of proteins. Under anaerobic conditions the protection factor was 13 in the presence of BSA, while with DMSO or t-butanol this factor was about 100 and with ethanol 300. In contrast to the low molecular weight OH scavengers oxygen enhanced radiation-induced double-strand breakage with BSA. It is assumed that protein peroxyl radicals may cause strand breakage.

Period Times and Helix Alignment during the Cyclic Migration of DNA in Electrophoresis Gels Studied with Fluorescence Microscopy

Fluorescence microscopy of YOYO-stained DNA molecules has been used to study the periodicity of the cyclic migration during constant field electrophoresis in agarose gels. The distribution of period times is asymmetric and wide, with a ratio of standard deviation to mean value of 0.50 for fields between 3 and 24 V/cm. The average period time decreases with the electric field strength, E, as E -1.2 , and increases with increasing molecular weight (38-740 kbp) and increasing gel concentration (0.5-2%). The period times for T2 DNA (164 kbp) are in good agreement with the time to the orientation undershoot, as measured in a separate linear dichroism study of YOYO-stained DNA undergoing ordinary zone electrophoresis (Carlsson et al., manuscript in preparation). This shows that linear dichroism can be used to quantify the period time of the cyclic motion and to investigate effects of changes in DNA or gel properties. The cyclic conformational changes in the global DNA conformation during migration have been characterized in terms of the apparent length L μ and end-to-end distance h μ of the molecular paths in the gel. The formation of U conformations dominates the cycle time, since it is slow compared to U slipping and retraction. The maximum and cycle-averaged orientation factors for the apparent molecular path are evaluated from the images to be 0.70 and 0.67 ± 0.03, respectively, for T2 DNA at 8 V/cm in 1% agarose. From these values the degree of helix orientation below the level of microscope resolution could be estimated by comparison with the total orientation factor for the DNA helix as measured with linear dichroism. The maximum and cycle-averaged orientation factor for the helix with respect to the global path were 0.27 and 0.22 ± 0.01, respectively. These orientation factor values demonstrate (1) a very high degree of field alignment of the global path and (2) a degree of stretching of the helix along that path which is considerable, and stronger than expected for a wormlike chain spanning a cavity with the average pore size. If the imaged path corresponds to the reptation tube, the estimated friction coefficient for motion along the tube is found to be close to the value in the buffer.

Recognition of Leuconostoc oenos strains by the use of DNA restriction profiles.

The chromosome of 41 Leuconostoc oenos strains obtained from collections in different countries was analysed with the aim of differentiating the strains. Pulsed field electrophoresis (TAFE) was used to separate large DNA fragments created by the restriction enzymes NotI, SfiI and ApaI, which specifically recognize guanines or cytosines. The genomic DNA of 11 strains was analysed initially with NotI and only four different restriction profiles were observed. The genome size ranged from 1.8 to 2.1 megabase pairs (Mbp). Constant field electrophoresis applied to DNA treatment with 19 different restriction enzymes showed that the size of the fragments obtained increased proportionally to the percentage G + C present at the site of restriction. EcoRI and HindIII profiles revealed that the zone between 9 and 23 kbp allowed differentiation of the strains tested. Thus, the 41 strains fell into 30 restriction groups using only two enzymes. Hybridization with a non-radioactive DNA probe coding for 16S rRNA revealed that there were two 16S genes on the chromosome.

6] Trapping electrophoresis of end-modified DNA in polyacrylamide gels

Field inversion gel electrophoresis (FIGE) dramatically improves the separation of large double-stranded (ds) DNA fragments (e.g., in the size range of yeast chromosomes) on agarose gels, as compared to constant field electrophoresis. However, relatively little improvement by FIGE has been found for short single-stranded (ss) DNA (hundreds of bases) on polyacrylamide gels. This chapter discusses the mechanism of a manyfold improvement in short ssDNA separation achieved by attaching a small protein, streptavidin, to one end of the DNA. The DNA orientation (elongation) during constant-field gel electrophoresis is probably what gives rise to the well-known phenomenon of “limiting mobility.” The essence of this phenomenon is that above a certain size of DNA, the mobility of the fragment no longer depends on its length and the electrophoresis loses its resolution. It should be noted that the whole phenomenon of the electrophoretic trapping of end-modified ssDNA has not yet been investigated thoroughly enough for practical purposes, in particular for DNA sequencing. Therefore, the chapter should perhaps be regarded as a guide for further experiments, rather than as an established standard for practical applications.

Pulsed field electrophoresis

AbstractPulsed field electrophoresis, or PFE, provides good separation between large molecules that under constant field electrophoresis are hard to isolate. This is due to the weak dependence of the constant field mobility on the molecular weight for these large molecules. If a spectrum of relaxation times exists that describes the recovery of the mobility to its constant field value after a reversal of the field, then we show that molecules with differing molecular weights are separated into two groups. Those with short relaxation times are unaffected by the cycling of the field and those with long relaxation times exhibit reduced mobilities. If the molecules adopt conformations that decrease their mobility initially, after a field reversal we demonstrate that a minimum develops in the mobility as a junction of the relaxation time. Using the model we demonstrate that effects of varying the switching times as a function of time. We predict that exponential rather than linear dependencies of the switching times on time increase the range of molecular weights over which enhanced separation can occur.