Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 19 recordsLinked to original sources

PubMed1992

DNA electrophoresis in uncross-linked polyacrylamide solution, studied by epifluorescence microscopy.

Electrophoresis of human DNA fragments (approximately 1 x 10(5) to 1 x 10(7) bases in size) was conducted in a solution of uncross-linked polyacrylamide contained in a horizontally mounted 1 mm diameter glass tube and monitored by epifluorescence microscopy. In presence of the polymer, molecular conformations described as a "trailing network" of DNA and a globular "head" were observed. The migration velocity varies between species differing in the size of the "head", and in the ratio between the size of the "head" and that of the trailing "network". By contrast, in pure buffer, lambda phage DNA migrates in a globular form at a mobility consistent with known macroscopic data. When electrophoresis in the polymer solution of an agarose plug preparation of Schizosaccharomyces pombe DNA was carried out after melting at 70 degrees C, a migrating DNA-agarose complex was observed. The complex was not fully dissociated by an agarose-hydrolyzing enzyme (Gelase).

Breast Neoplasms↗

PubMed1992

Resolution of circular, nicked circular and linear DNA, 4.4 kb in length, by electrophoresis in polyacrylamide solutions.

Circular DNA of more than 1,400 bp in size is known not to migrate into polyacrylamide gels. The migration of supercoiled plasmid pBR322 DNA (4,363 by) into uncrosslinked polyacrylamide (Mw 5 x 10(6)) solutions and its separation, on the basis of conformation, from its nicked form is demonstrated in this study. Migration of the supercoiled, nicked circular and linear forms of the plasmid DNA is retarded in proportion to the concentration of uncrosslinked polyacrylamide, the degree of retardation being highest for the nicked circular form. Decreasing the level of supercoiling of the covalently closed circular form by decreasing the concentration of the intercalating dye (ethidium homodimer) shows that the degree of retardation decreases in proportion to the superhelix density.

DNA, Bacterial↗

PubMed1992

Concave Ferguson plots of DNA fragments and convex Ferguson plots of bacteriophages: evaluation of molecular and fiber properties, using desktop computers.

A desktop computer program evaluating physical properties of DNA and bacteriophages is presented. The analysis is based on data obtained from capillary and submarine-type agarose electrophoresis. Native molecular/particle properties and properties of the gel (or polymer) medium can be derived from electrophoresis at several gel concentrations. This is done conveniently by a computerized evaluation of the semi-logarithmic plot of mobility vs. gel concentration, designated the Ferguson plot. In application to most proteins, this plot is linear and computer programs exist to evaluate it. However, nonlinear Ferguson plots have assumed great importance in view of the fact that the plots are concave for DNA. Similarly, convex plots are important since they prevail in the electrophoresis of large particles in agarose. The computer program reported here is the first to (i) address concave Ferguson plots and (ii) allow for the evaluation of both cases using a desktop computer. Program ELPHOFIT version 2.0, a Macintosh application, is available upon request.

Bacteriophages↗

PubMed1992

Distinction between supercoiled and linear DNA in transverse agarose pore gradient gel electrophoresis.

Four species of linear DNA and the first four members of a linking series, generated by treatment of plasmid DNA (PUC19, 2.7 kb) with mitochondrial topoisomerase I, were differentiated by transverse agarose pore gradient gel electrophoresis. The experimental curves of migration distance vs. agarose concentration (Ferguson curves) of supercoiled DNA exhibit a steeper trajectory than those of linear DNA of the same size range. As a consequence, the four supercoiled species exhibit an increase in apparent size (relative to linear DNA standards) with increasing agarose concentration. Both the crossing of the Ferguson curves with those of linear standards as well as the apparent size increase with agarose concentration can serve to detect supercoiled plasmid-sized DNA in mixtures with linear DNA.

DNA↗

PubMed1992

Computer-aided analysis of DNA curves on transverse gradient gels.

Transverse pore gradient polyacrylamide gel electrophoresis of DNA restriction fragments was used to generate gel patterns describing migration distance as a function of gel concentration (Ferguson curves). These Ferguson curves were digitized, traced and analyzed with the aid of a personal computer. The traced curves were plotted semi-logarithmically and the plots were subjected to least-squares linear regression analysis to yield values of the slope (KR) and the intercept at %T = 0 (YO). These values are highly precise since they are based on approx. 100 measurements per curve. The computerized method reduces the errors due to manual measurements of migration distances and is time and labor saving. The method is still limited to intra-experimental comparison of Ferguson curves, since it does not as yet comprise a determination of gel concentration. At present, curve tracing remains semi-automated, requiring manual intervention when Ferguson curves cross or approach one another. Potentially, the importance of the computerized analysis of transverse pore gradient gels lies in the rapid quantitative interpretation of Ferguson curves for detection of anomalously migrating DNA species. Potentially, that application provides a more sensitive and informative mode of detection than either the mere visual observation of crossing Ferguson curves or of a shift in mobility at a single gel concentration.

Buffers↗

PubMed1992

Transverse agarose pore gradient gel electrophoresis of DNA.

Transverse agarose pore gradient gels were prepared on GelBond in the concentration range of nominally 0.2-1.5% SeaKem GTG agarose, using density stabilization by glycerol and incorporation of a dye to define the gel concentration at each point on the pore gradient gel. The distribution of the dye was evaluated by photography, video-acquisition and digitization of the gradient mixture and by densitometry of the gel. The gel was applied to the electrophoresis of a 1-kb standard ladder of DNA fragments, using standard submarine apparatus. The method extends to agarose gel electrophoresis the benefits of semi-automated analysis of 'Ferguson curves' described in application to polyacrylamide gel by Wheeler et al. (J. Biochem. Biophys. Methods 24, 171-180).

Coloring Agents↗

PubMed1991

Size separation of polystyrene sulfate particles (189 to 1085 nm radius) in solutions of methyl-hydroxypropyl-cellulose of different chain lengths.

Polystyrene sulfate particles of 189 to 1,085 nm radius are retarded in their electrophoretic migration in solutions of methyl-hydroxypropyl-cellulose (4,000 and 15,000 cP viscosity for a 2% solution). The retardation is proportional to polymer concentration and increases inversely with particle size. This suggests a separation mechanism based on size-dependent exclusion from the polymer network, as found previously in other liquid polymer media. In the size range of approximately 200 to 600 nm radius, the 4,000 cP polymer exhibits a steeper decrease of retardation with particle size than the 15,000 cP polymer, but fails to exhibit any size dependent retardation beyond that range.

Cholic Acids↗

PubMed1991

Electrophoretic separation of S. pombe chromosomes in polyacrylamide solutions using a constant field.

Previous electrophoretic separations of megabase (Mb) sized DNA have been achieved in pulsed electric fields, using agarose gel as a matrix. The present study demonstrates separations of Mb sized DNA due to a retardation of migration in proportion to the concentration of uncrosslinked polyacrylamide of 5 x 10(6) molecular weight, using a constant electric field. Potentially, the method should be applicable to large DNA in general, greatly reducing the instrumental complexity of such separations and rendering them compatible with capillary electrophoresis apparatus.

Chromosomes, Fungal↗

PubMed1991

Apparatus for gel electrophoresis with continuous monitoring of individual DNA molecules by video epifluorescence microscopy.

Apparatus for gel electrophoresis with continuous scanning by video epifluorescence microscopy was constructed and applied to measurements on individual DNA molecules in the size range of several hundred kilobase pairs. The gel electrophoresis apparatus comprises a horizontal glass cell and several commercially available components--an epifluorescence microscope, an image intensifier, two video cameras, a monitor, videotape recorder and voltmeter. Data in the form of videotapes can be subsequently analyzed on a computer graphics work station.

DNA↗

PubMed1991

Conformational correlatives of DNA band compression and bidirectional migration during field inversion gel electrophoresis, detected by quantitative video epifluorescence microscopy.

Individual DNA molecules in the Mb size range were monitored by epifluorescence video microscopy during field inversion gel electrophoresis (FIGE). DNA migrating in an agarose gel gives rise to characteristic V-conformational elements and when doing so exhibits a reduced mobility. When the V-conformational elements per DNA molecule are few, the degree of retardation appears proportional to the number of V's, and since larger DNA species exhibit more V's, to DNA size. For a particular pulse frequency, the proportionality breaks down progressively as the number of V-conformational elements per DNA molecule increases. The loss of proportionality between DNA length and migration rate is being correlated with the macroscopically observed loss of electrophoretic size discrimination known as band compression. For a particular pulsing frequency and size class of DNA, the loss of size discrimination is thought to be due to the different orientations of migration, caused by the asymmetric distribution of V-conformational elements when the number of these elements is moderate. Small and very large DNA by contrast migrate with the direction of the biased field. These events, analyzed by microscopic measurement, are consistent with the known macroscopically observed double-valued mobilities in FIGE.

DNA↗

PubMed1991

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.

Bacterial Outer Membrane Proteins↗

PubMed1991

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments.

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamide gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis, using a discontinuous buffer system. Polyacrylamide gels at pH 8.9, 2 degrees C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 micrograms/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72-23130 bp, while agarose gels at pH 8.5, 25 degrees C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 microgram/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 x TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 x TBE buffer, with a substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification by Rf, the migration distance relative to a sharply defined "buffer front" (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1991

Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system.

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE)(15%CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4 degrees C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3% T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest % T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3% T have not as yet been measured. As previously suggested, the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched, "reptating" DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5% T when 15% DATD rather than 2.5% Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4 degrees C to 25 and 50 degrees C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a "reptating" to a randomly distributed molecule, due to increased Joule heat.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗

PubMed1991

Free mobility determination by electrophoresis in polyacrylamide containing agarose at a nonrestrictive concentration.

In the determination of the free mobility, related to the surface net charge, by quantitative gel electrophoresis, the previous arbitrary extrapolation of Ferguson plots from the lowest gel concentrations that give a mechanically stable gel to 0% T has recently been replaced by measurement of mobilities across that concentration range, using the addition of 0.5% agarose to polyacrylamide at the various low concentrations in application to a DNA fragment 155 bp in size (Orbán, L. et al., in preparation). The present study applies that approach to several proteins and DNA fragments smaller than 1300 bp, using 0.4% agarose in polyacrylamide gels of varying concentration. The intercepts of the plots with the mobility axis provide experimental data by which the free mobility in polyacrylamide gel electrophoresis can be estimated for molecules not significantly retarded in their migration at the agarose concentration admixed to polyacrylamide. Across the gel concentration range below 3% T, in the presence of agarose, the Ferguson plots of proteins and DNA fragments are convex. It was shown by mass spectrometry that this convex curvature of the plots in the mixed polymer is not significantly due to low polymerization efficiency in the concentration range of liquid polyacrylamide (below 3%T).

Chemical Phenomena↗

PubMed1991

Gel electrophoresis of polystyrene particles in glutaraldehyde crosslinked polyvinyl alcohol.

Polystyrene sulfate and carboxylate particles (19-189 nm radius) were subjected to electrophoresis in glutaraldehyde crosslinked polyvinyl alcohol of molecular weight 25.000 and 650.000 Da at various concentrations. The degree of crosslinking is severely limited by the mechanical properties of the gels that deteriorate beyond a glutaraldehyde concentration which decreases with increasing polyvinyl alcohol chain length. The effective fiber radius of the short-chain and long-chain polymer fiber was 45 +/- 25 and 131 +/- 47 nm, respectively. Thus, these media do not significantly exceed the apparent fiber thickness of agarose, are more difficult to prepare--but are well-defined synthetic products rather than natural ones, and have the advantage of carrying no net charge and can therefore be expected to exhibit no electroendosmosis.

Cross-Linking Reagents↗

PubMed1991

Detection of conformational and net charge differences in DNA-protein complexes by quantitative electrophoresis on polyacrylamide-agarose copolymer gels.

The Galactosidase repressor (GalR) of Escherichia coli modulates the expression of the gal operon by binding to two DNA operators, OE and O1. The OE and O1 elements are 16 bp pallindromic DNA sequences, differing in four of the base pairs. OE and O1 DNA fragments, both free and complexed with repressor, were analyzed by "quantitative gel electrophoresis". By the criteria of that method, applied to the linear Ferguson plots of both DNA fragments and the linear ranges of those of the DNA-GalR complexes, it was shown that the apparent size of DNA increases upon repressor binding. Moreover, this size increase is greater for the complex with the O1 operator than for the complex with the OE operator in the case that GalR is located in the center of a 155 bp DNA fragment. This is not the case when GalR is located in a peripheral position. By contrast with their size differences, the centrally located GalR-O1 and GalR-OE complexes appear to possess indistinguishable net surface charge densities as judged from the intercepts with the mobility axis. The larger size of the complex with centrally located O1 fragment, as compared with that bearing the OE fragment, is interpreted as being due to bending of the DNA-protein complex, since an authentically bent fragment of a plasmid with bent upstream activator sequence also exhibits a larger slope of the Ferguson plot, and thus the larger size, than predicted on the basis of its DNA chain length (bp).(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗

PubMed1991

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).

DNA, Circular↗

PubMed1991

Electrophoresis with intermittent scanning of the migration path: detection of resolution within shortened time.

Intermittent optical scanning (by detection of optical density or fluorescence) of the electrophoretic migration path was applied to the resolution of two dyes under an arbitrary set of conditions. Scanning at 5-min intervals allows for detection of resolution between the two zones at least 3 times faster than conventional automatic zone detection employing a detector at the end of the migration path. This result promises that replacing stationary by mobile detectors in general would result in a substantial time saving for automated detection of electrophoretic zones.

Densitometry↗