The free solution mobility of DNA

The free solution mobility of DNA has been measured by capillary electrophoresis in the two buffers most commonly used for DNA gel electrophoresis, Tris-borate-EDTA (TBE) and Tris-acetate-EDTA (TAE). The capillaries were coated with polymers of either of two novel acrylamide monomers, N-acryloylaminoethoxyethanol or N-acryloylaminopropanol, both of which are stable at basic pH and effectively eliminate the electroendosmotic mobility due to the capillary walls. The free solution mobility of DNA in TAE buffer was found to be (3.75 +/- 0.04) x 10(-4) cm2 V-1 s-1 at 25 degrees C, independent of DNA concentration, sample size, electric field strength, and capillary coating, and in good agreement with other values in the literature. The free solution mobility was independent of DNA molecular weight from approximately 400 base pairs to 48.5 kilobase pairs, but decreased monotonically with decreasing molecular weight for smaller fragments. Surprisingly, the free solution mobility of DNA in TBE buffer was found to be (4.5 +/- 0.1) x 10(-4) cm2 V-1 s-1, about 20% larger than observed in TAE buffer, presumably because of the formation of nonspecific borate-deoxyribose complexes.     

SDS agarose gels for analysis of proteins

A new agarose-based protein electrophoresis gel system is described. The system consists of a highly resolving agarose, MetaPhor XR (FMC BioProducts, Rockland, ME, USA) dissolved in urea and TBE buffer and a stacking gel composed of a high gel-strength agarose, SeaKem Gold (FMC BioProducts). TBE containing sodium dodecyl sulfate (SDS) is used as electrophoresis buffer. The disadvantages of traditional agarose gels have been overcome, and several advantages over polyacrylamide gels have been demonstrated. The system is capable of high-resolution separation of small proteins and has a dynamic separation range equivalent to a 4%-20% gradient polyacrylamide gel. Furthermore, the staining of protein bands by Coomassie Brilliant Blue is very uniform in this gel, and depending on the protein, higher detection sensitivity can be obtained compared to SDS polyacrylamide gels. In Western blotting, proteins are more efficiently transferred to the membrane from the agarose gel than from polyacrylamide gels. Finally, the exceptional stability of agarose allows for gels to be precast and stored for a year.     

New substrates of papain, based on the conserved sequence of natural inhibitors of the cystatin family

A discontinuous borate/formate buffer system is presented for horizontal polyacrylamide gel electrophoresis of DNA fragments. The resolution potential of the system could be altered by changing the total monomer concentration (5-9%T), the concentration of the crosslinker piperazine diacrylamide (2-5%CPDA), as well as the concentration of formate in the gel (40-120 mM), the leading ion of the buffer system. The separation of DNA fragments would be improved by increasing the migration distance from 22 to 28 cm. This discontinuous polyacrylamide gel electrophoresis system proved highly reproducible.     

Normal and anomalous electrophoretic behavior of polymerase chain reaction-based DNA polymorphisms in polyacrylamide gels

The electrophoretic mobility of five single tandem repeats (STRs) and four amplified fragment length polymorphisms (AFLPs) in polyacrylamide gels was tested under denaturing and nondenaturing conditions. Relative anomalous mobility in nondenaturing conditions was found in one AT-rich AFLP (3'ApoB) and in two AT-rich STRs (HUMACTBP2 and HUMF13A1). In these cases, highly anomalous electrophoretic behavior was found even when changes were made in the %T value alone. In such cases typing results were affected by the gel composition. The mobility of these systems was nevertheless normal under denaturing conditions. As a consequence of this study, we recommended that the typing of these systems should only be performed under denaturing conditions and we also recommend the further study of the electrophoretic behavior of repetitive DNA polymorphisms, especially AT-rich systems, before they are used in forensic or genetic applications.     

Simple miniaturized gel system for DNA sequence analysis

A simple miniaturized gel system suitable for DNA sequencing is described. Small ultrathin polyacrylamide gels are cast, eight or more at a time, using standard microscope slides. Gels, ready to use, can be stored for approximately 2 weeks. Gels are run horizontally in a standard mini-agarose gel apparatus. Typical run times are 6-8 min. A novel sample loading system permits volumes of standard sequencing reactions as small as 0.1 microl to be analyzed. Sequencing ladders were visualized using 35S-labeled DNA by autoradiography and by colorimetric detection. Band resolution compares favorably with that of large gels. The methods introduced here serve as a step toward the miniaturization of DNA sequencing and are amenable to automated sample loading and detection.     

The resolution between two native proteins and between their sodium dodecyl sulfate-complexes in agarose and polyacrylamide gel electrophoresis

Commercial gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path (HPGE-1000 apparatus, LabIntelligence) makes it possible to measure band width and migration distance as a function of the duration of electrophoresis. As a result, resolution can be evaluated quantitatively and therefore different gel media can be compared objectively. The resolution of fluorescein carboxylate labeled conalbumin (molecular mass 86 kDa) and soybean trypsin inhibitor (22.7 kDa) in gel electrophoresis was found to increase as a function of the gel type in the order SeaKem GTG-, SeaKem Gold-agarose, 2% N,N'-methylenebisacrylamide cross-linked polyacrylamide, MetaPhor-XR-, and SeaPrep-agarose. The advantage in resolving capacity of SeaPrep agarose over the polyacrylamide gel was by a factor of up to five. The resolving capacity of the agaroses was in indirect relation to the degree of electroendosmosis. In all media, resolution increased with migration distance (time). The same proteins when reacted with sodium dodecyl sulfate (SDS) resolve (i) better at up to 6% SeaPrep agarose concentration than in polyacrylamide, as in the gel electrophoresis of the native proteins; (ii) less effectively, by contrast, at SeaPrep agarose concentrations > 6%, than in polyacrylamide gel; and (iii) significantly better in 4-6% SeaPrep agarose than in 4-6% SeaKem GTG agarose. Since Ferguson plot analysis in both agarose and polyacrylamide gels shows that the two SDS-proteins are larger than the native proteins with which they are complexed, the superiority of polyacrylamide gels above 7% appears to be correlated with the fact that its mean pore radius, estimated for both media using identical assumptions and identical rigid spherical standards - proteins, is approximately seven times larger than that of SeaPrep agarose in the concentration range of 3-8%, and that therefore the molecular "fit" in polyacrylamide is closer than that in SeaPrep agarose of the concentration range used. The dependence of resolution on the ratio of particle radius to mean pore radius ("fit") is also suggested by the fact that the two SDS-proteins resolve in a biphasic dependence on gel concentration in both agarose and polyacrylamide, with a maximum at 6% agarose and 10% polyacrylamide.     

Effects of supercoiling in electrophoretic trapping of circular DNA in polyacrylamide gels

Electrophoretic velocity and orientation have been used to study the electric-field-induced trapping of supercoiled and relaxed circular DNA (2926 and 5386 bp) in polyacrylamide gels (5% T, 3.3% C) at 7.5-22.5 V/cm, using as controls linear molecules of either the same contour length or the same radius of gyration. The circle-specific trapping is reversible. From the duration of the reverse pulse needed to detrap the molecules, the average trap depth is estimated to be 90 A, which is consistent with the molecular charge and the field strengths needed to keep molecules trapped. Trapped circles exhibit a strong field alignment compared to the linear form, and there is a good correlation between the enhanced field alignment for the circles and the onset of trapping in both constant and pulsed fields. The circles do not exhibit the orientation overshoot response to a field pulse seen with linear DNA, and the rate of orientation growth scales as E(-2+/-0.1) with the field, as opposed to E(-1.1+/-0.1) for the linear form. These results show that the linear form migrates by cyclic reptation, whereas the circles most likely are trapped by impalement on gel fibers. This proposal is supported by very similar velocity and orientation behavior of circular DNA in agarose gels, where impalement has been deemed more likely because of stiffer gel fibers. The trapping efficiency is sensitive to DNA topology, as expected for impalement. In polyacrylamide the supercoiled form (superhelical density sigma = -0.05) has a two- to fourfold lower probability of trapping than the corresponding relaxed species, whereas in agarose gels the supercoiled form is not trapped at all. These results are consistent with existing data on the average holes in the plectonemic supercoiled structures and the fiber thicknesses in the two gel types. On the basis of the topology effect, it is argued that impalement during pulsed-field electrophoresis in polyacrylamide gels may be useful for the separation of more intricate DNA structures such as knots. The results also indicate that linear dichroism on field-aligned molecules can be used to measure the supercoiling angle, if relaxed DNA circles are used as controls for the global degree of orientation.     

Migration of single-stranded DNA in polyacrylamide gels during electrophoresis

By using fluorescence recovery after photobleaching (FRAP) and electric birefringence, the migration of single-stranded DNA in polyacrylamide gels and orientation as a response to an electric pulse were investigated. Electrophoretic mobility is in good agreement with the model of biased reptation including fluctuations. The determination of the electrophoretic mobility in solution, mu0, allows an estimation of the gel pore diameter seen by the molecule. As previously observed for double-stranded DNA, the electric birefringence results from two processes: the alignment of the molecule along the electric field and the elongation of the primitive path in the gel, for long single-stranded DNA (>2000 bases). The combination of results obtained with the two techniques allows us to propose experimental conditions to improve the separation of single-stranded DNA with pulsed field techniques.     

Postprandial lipemic response is modified by the polymorphism at codon 54 of the fatty acid-binding protein 2 gene

Agarose gels have been studied by atomic force microscopy (AFM). The experiments were especially designed to work in aqueous conditions, allowing direct observation of the "unperturbed" gel without invasive treatment. AFM images clearly show strong dependence of pore diameter and its distribution on ionic strength of the solvent. As the ionic strength increases, the distribution becomes broader and the position of its maximum shifts toward higher values. The evolution of the distribution curves indicates that gels become more homogeneous with decreasing Tris-borate-EDTA (TBE) buffer concentration. An empirical law of the mean pore diameter as a function of the ionic strength is established. In agreement with our previous work we found that, for a given ionic strength, the pore diameter increases when the agarose concentration decreases and that the wide pore diameter distribution narrows as the gel concentration increases.     

Small circles of helical DNA obtained on the basis of transcriptional pause sites sequence

The 21-base pair synthetic DNA duplexes with basic 'pause-motif site ('CATGC') were ligated head-to-tail to produce linear and circular multimers. This also was done from other closely related sequences. Electrophoretic mobilities of the linear multimers in polyacrylamide gels were determined under the standard and modified conditions. We revealed that small linear multimers (approximately 90 bp) were characterized by comparable value of gel retardation relative to the well known curved DNA, while longer multimers (130 to approximately 170 bp) had only slightly expressed mobility anomaly. Nevertheless these multimers containing nontruncated 'pause-motif were capable of cyclization, in particular, formation of unusually small circles while truncated ones were not. We conclude that basic 'pause-motif site increases the closure ability while the multimers based on truncated 'pause motif fail to curve into the small circles. We tend to explain this situation as a result of intrinsic bending as well as the influence of the thermal fluctuations of DNA, the latter most probably can be associated with 'pause motif'. We have estimated the equilibrial and maximal bend angles per 10.5 bp to be 12 degrees to approximately 16 degrees and 32 degrees accordingly under experimental conditions of our study.     

Allophycocyanin from the filamentous cyanophyte, Phormidium luridum

Allophycocyanin from the filamentous cyanophyte, Phormidium luridum, was purified by ammonium sulfate fractionation and ion exchange chromatography on brushite columns. The specific absorption coefficient (E 0.1% 1cm) of purified allophycocyanin was 6.1 in distilled water and 7.3 in 0.05 M potassium phosphate buffer (pH 7). Absorption maxima of allophycocyanin occurred at 650, 618 (shoulder), 350, and 275 nm. Circular dichroic spectra displayed positive ellipticity bands at 655 and 625 nm, and a major negative ellipticity band at 340 nm. Computer analysis of the circular dichroic spectrum of allophycocyanin from 207 to 243 nm indicated that the secondary structure contained 60% alpha helix and 40% beta form. The estimated molecular weight of allophycocyanin on Sephadex G-200 columns at pH 7.0 was 155,000. Electrophoretic examination of allophycocyanin on sodium dodecyl sulfate polyacrylamide gels revealed two subunits, alpha and beta, with apparent molecular weights of 17,300 and 19,000, respectively. Densitometric analysis of unstained gels at 600 nm indicated that one phycocyanobilin chromophore was associated with each subunit. Treatment of allophycocyanin with 12% formic acid or 8 M urea and subsequent removal of the denaturant yielded a derivative with spectroscopic characteristics similar to phycocyanin. Subsequent incubation in phosphate buffer (pH 7), but not in acetate buffer (pH 5) or in water, was accompanied by a progressive reappearance of absorption maxima at 650 and 618 nm (shoulder), and positive ellipticity bands at 655 and 617 nm. Automated sequence analysis of allophycocyanin (a) showed that the sequence of amino acids at the amino terminus of the alpha and beta subunits is different, (b) showed that the subunits occur in a ratio of 1:1, and (c) demonstrated sequence homology at the amino terminus of allophycocyanin, phycocyanin, and phycoerythrin.     

A simplified polyacrylamide gel electrophoresis apparatus for simultaneous application of multiple buffer systems or detergent combinations

A previous design of an apparatus for the simultaneous fractionation by polyacrylamide gel electrophoresis in 10 different buffer systems (1) was replaced by a greatly simplified new design, employing small, cylindrical buffer partitions within the lower buffer reservoir and/or upper buffer reservoir of a conventional, temperature-regulated polyacrylamide gel electrophoresis apparatus for cylindrical gels. The apparatus was tested in application to the problem of simultaneous polyacrylamide gel electrophoresis in different buffer systems with the purpose of optimizing the operative pH for a particular fractionation problem. It was also applied to fractionations in a single buffer system to which various combinations of ionic and nonionic detergents were admixed.     

Rat intestinal brush border membrane peptidases. I. Solubilization, purification, and physicochemical properties of two different forms of the enzyme

Two brush border peptidases have been isolated from the particulate fraction of the rat intestinal mucosa and purified to homogeneity as judged by polyacrylamide gel electrophoresis, starch gel electrophoresis, isoelectric focusing, and double immunodiffusion. For convenience, the peptidases have been designated peptidase F (fast) and S (slow) on the basis of their anodic mobilities. The isoelectric point of peptidase F was 4.76 and of peptidase S, 5.10. Both enzymes are glycoproteins. The amino acid compositions of the two peptidases are similar. The same carbohydrates are found in both enzymes, but there are differences in the molar concentrations of individual sugars. Peptidase S has greater concentrations of mannose and galactose and of hexosamines than peptidase F, while sialic acid is slightly greater in peptidase F. Carbohydrate accounted for approximately 19% and 23% of the weight of peptidases F and S, respectively. Estimates of the molecular weights of both enzymes by gel filtration gave values of 280,000. Electrophoresis of the enzymes under denaturing conditions on sodium dodecyl sulfate polyacrylamide gels indicated that each enzyme is a dimer consisting of two subunits of equal molecular weight, 140,000.     

Helical periodicity of GA-alternating triple-stranded DNA

Homopurine-homopyrimidine tracts (18 or 28 bp) containing predominantly GA-alternating sequences were inserted between two bent DNA loci composed of six (dA)6.(dT)6 repeats. For each homopurine tract, the DNA length between the bent DNA loci was varied by one base pair over a full helical turn. The two series of bent DNA fragments were electrophoresed in 5% polyacrylamide gel to measure the gel mobilities, which reflected the overall extent of DNA bending of each DNA fragment. By comparing the gel mobilities between the two series of bent DNA fragments, the helical periodicity of the GA-alternating duplex was determined to be 10.4 bp/turn. The two series of bent DNA fragments were also electrophoresed upon formation of the intermolecular triplex at the homopurine.homopyrimidine tracts. Comparison of the gel mobilities between the two series of DNA fragments showed that the helical periodicity of triplexed DNA of GA-alternating sequence was 11.2 bp/turn. Triplexed DNA with homopurine and homopyrimidine oligodeoxyribonucleotides had the same helical periodicity, while hybrid triplex DNA associated with homopyrimidine oligoribonucleotide had a slightly more wound structure with 11.1 bp/turn.     

An exactly solvable Ogston model of gel electrophoresis IV: sieving through periodic three-dimensional gels

In this article, we extend our recently developed lattice model of gel electrophoresis to periodic three-dimensional gels made of either isolated obstacles or infinitely long fibers. Exact mobilities are calculated using a much improved numerical method that allows us to treat very large systems. A comparison of the exact mobilities and free available volumes indicates that the main assumption of the Ogston-Morris-Rodbard-Chrambach model (OMRCM), which postulates that the mobility (mu) of charged particles is directly related to the fractional gel volume available to them, is not valid. However, a study of the gel concentration and analyte size dependence of the zero-field mobility indicates that the OMRCM and the Ferguson plots can indeed be used to obtain useful, semi-quantitative information about the gel properties. A procedure to study more realistic three-dimensional gel systems is discussed.     

Gradient polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate: a practical approach to muscle contractile and regulatory proteins

Two gradient systems for polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) are described, with emphasis on improvements accumulated over two decades of studies on contractile proteins and regulatory enzymes from smooth muscle. The first "big slab" system utilizes 18 x 20 x 0.1 cm3 gels and a 10-18% acrylamide gradient, optimized for a high resolution of 10 to 500 kDa polypeptides. Eight (or more) gels are cast simultaneously with a gradient formation from "bottom to top" and 20% glycerol is added to the 18% acrylamide solution. The second "minislab" system represents an improved version of the system of Matsudaira and Burgess (Anal. Biochem. 1978, 87, 386-396), with 8 x 10 x 0.05 cm3 gels and 5-15% or 9-18% acrylamide gradient ranges. They are cast from "top to bottom" in 28-piece batches also with the addition of glycerol for improved gradient formation. Both types of gels can also be cast individually using a specially designed pestle-type gradient maker. For gel destaining, a convenient continuous hydrodynamic destainer is also described.     

Occupation and breast cancer mortality in a prospective cohort of US women

We report the isolation of the functional form of the Ca-ATPase from porcine cardiac sarcoplasmic reticulum (SR) membranes, taking advantage of the ability of this enzyme to bind to the nucleotide site affinity dye, Reactive Red 120. Conditions that optimize the solubility and functional stability of the cardiac Ca-ATPase in detergent during the purification procedure are essential to its recovery. The purified Ca-ATPase migrates as a single band on Coomassie blue-stained polyacrylamide gels and exhibits high specific activity (2.5 IU at 25 degreesC) and functional stability. Similar enrichment of the Ca-ATPase estimated from either relative amounts of the 100-kDa protein band on polyacrylamide gels or steady-state concentrations of phosphorylated enzyme intermediate (E-P) demonstrate that neither nonfunctional Ca-ATPases nor non-Ca-ATPase proteins migrating with an apparent molecular weight of 100 kDa constitute a significant fraction of these preparations. Steady-state levels of E-P are 1.3 and 8.6 nmol/mg protein, respectively, for native cardiac SR membranes and the final purified fraction. These values, in comparison to the maximum value (9.1 nmol/mg) for the 110-kDa protein, agree well with estimates of total Ca-ATPase abundance from gel densitometry for both preparations and indicate full site reactivity, i.e., one phosphorylation site for each 110-kDa cardiac Ca-ATPase polypeptide chain.     

Two-dimensional separations: capillary electrophoresis coupled to channel gel electrophoresis

Two-dimensional separations provide extremely high peak capacities. Coupling capillary zone electrophoresis with ultrathin channel gel electrophoresis offers a convenient and efficient way to perform such two-dimensional microseparations. By means of in situ polymerization, high-concentration (up to 50%T) polyacrylamide gels are prepared in 75 mm long, 25 mm wide, and 40 microns thick rectangular channels. By moving the outlet end of the capillary electrophoresis capillary across the entrance of the channel, both separations are completely preserved. Mixtures of peptides labeled by fluorescein isothiocyanate (FITC) are well resolved in less than 15 min, with theoretical plate numbers in the range of 20,000-50,000 for each independent separation. Significant enhancement in separation efficiency and peak capacity over one-dimensional separations are demonstrated by this combination. The two-dimensional separations of a model mixture of peptides, a tryptic digest of trypsinogen, and < 0.05% of an individual B2 neuron from the marine mollusk Aplysia californica are presented.     

Electrophoretic behavior and size distribution of the acidic polysaccharides produced by the bacteria Bradyrhizobium (Chamaecytisus) strain BGA-1 and Bradyrhizobium japonicum USDA 110

The electrophoretic behavior in polyacrylamide gels of the acidic polysaccharides produced by the soil bacteria Bradyrhizobium (Chamaecytisus) strain BGA1 and Bradyrhizobiumjaponicum USDA1 10 has been studied. Both polysaccharides were polydisperse, producing a ladder-like pattern after fixation with Alcian Blue and silver staining of the gel. The polysaccharide molecules were separated according to their size, and they behaved as a collection of flexible random coils of different size and similar charge/mass ratio. The electrophoretic behavior was not affected by the presence of acetyl groups in the polysaccharide. The range of molecular weights of the exopolysaccharide produced by B. japonicum USDA110 was wider and with larger molecules than that of the polysaccharide produced by strain BGA1. The resolution was dependent on the electrophoresis buffer; the best results were achieved with Tris-borate; in Tris-glycine buffer, the resolution was worse, and it was not improved by the addition of sodium dodecyl sulfate (SDS).     

Satellite DNA and heterochromatin of the flour beetle Tribolium confusum

The chromosomes of Tribolium confusum have conspicuous bulks of pericentromeric constitutive heterochromatin. The amount of heterochromatin measured by C-banding in metaphase chromosomes is estimated to be 40-45%. It is composed of an A + T rich DNA according to the distamycin A/diamidinophenylindol staining of chromosomes. Restriction analysis of isolated T. confusum genomic DNA shows that this species has a satellite DNA that constitutes about 40% of the genome. Cloning and sequencing experiments reveal a monomer length of 158 base pairs and a copy number of 5.77 x 10(5) per haploid genome. Its sequence is A + T rich (73%), with direct and inverted repeats, one of them with a possibility of forming stable cruciform structure. The abundance, monomer length, and the mutation rate are similar to those found in other satellite families from different species of Tenebrionidae, but no sequence homology has been found among them. No retarded mobility of satellite DNA, characteristic for molecules with sequence-induced curvature, has been detected by electrophoresis on nondenaturing polyacrylamide gels. In situ digestions with restriction enzymes and in situ hybridization show that this satellite DNA is located in pericentromeric positions of all chromosomes coinciding with C-bands.