Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 73 records · Page 4Linked to original sources

PubMed1995

Computer simulation of the directional displacement of rod-shaped, arc-shaped, and circular objects in an array of obstacles, representing a simple model for the gel electrophoresis of small DNA.

The gel electrophoresis of DNA of identical length but various static conformations was simulated using a two-dimensional model of the movement of rod-shaped, arc-shaped, and circular objects through random arrays of disk-shaped obstacles. At low obstacle density, the displacement rate of these objects decreases from the rod-shaped to the circular to the arc-shaped objects. At high obstacle densities, the displacement rate of circular objects approaches zero. The alignment of the arc-shaped objects along the axis of the directional movement of the objects were retarded in their movement by collisions with the obstacles; the number of collisions of the former, in view of their greater ability to align, was less than that of the latter. Circular objects were exclusively retarded by collisions, while the arc-shaped objects exhibited an additional retarding mechanism, viz. the suspension ("hanging") on the obstacles. When the rigid objects were made flexible, their displacement increased. The increase was most pronounced with the circular objects, allowing them to penetrate at obstacle densities from which the rigid objects were excluded.

Computer Simulation↗

PubMed1995

Commercial automated gel electrophoresis apparatus: application to DNA, band dispersion, nonlinear Ferguson curves, and isolation.

Recently available commercial automated gel electrophoresis apparatus with intermittent scanning of fluorescently labeled gel patterns (the HPGE-1000 apparatus of LabIntelligence, Menlo Park CA) was tested with regard to (i) its applicability to DNA in its native conformation, (ii) its ability to recognize the correct number of components, (iii) its capability to evaluate the width and shape of bands detected during electrophoresis, (iv) its ability to yield nonlinear Ferguson plots in a labor-saving fashion, and (v) its preparative potential. Ethidium homodimer (EtD) DNA (bp) ratios were systematically varied and the mobility of DNA fragments labeled at each ratio was measured in order to find a ratio which provided an unaltered mobility and presumably therefore an unaltered conformation of the fragment. That ratio was found to be 1/40 EtD/DNA (bp) or less. With such weak labeling of DNA, a representative fragment of 527 bp length requires a minimum load of 200 ng and a 2 micrograms load for a full-scale peak height. Using the baseline automatically selected by the software of the apparatus, the band areas of the 17 components of a DNA digest were consistently evaluated by the software, as evidenced by the proportionality between DNA length and area. The areas of the separated bands of DNA fragments of 1857 and 121 bp length were found to be constant with time of electrophoresis. The dispersion coefficient was found to decrease with agarose concentration in electrophoresis at 1 V/cm; however, at higher field strength, the band width of the 1857 bp fragment was surprisingly found to increase with gel concentration, presumably due to stretching.(ABSTRACT TRUNCATED AT 250 WORDS)

Buffers↗

PubMed1995

Dispersion coefficients of a protein and DNA fragment in polyacrylamide gel electrophoresis as a function of parameters defining the effective gel pore size and particle size.

The dispersion coefficient, D', of the representative homogeneous protein, conalbumin, decreases linearly as the polyacrylamide concentration increases from 4 to 14%T (2%C), and varies in a biphasic fashion as %C (Bis) is increased from 2 to 20%, with a broad peak between 5 and 15%C. D' increases linearly with the concentration of the initiator, potassium persulfate, in the range of 0.01-0.15%. D' remains constant when the field strength is varied from 5 to 15 V/cm. A DNA fragment (1857 bp) exhibits a constant D' in 4-6% polyacrylamide (2%C) at a field strength of 1 V/cm, and a linearly increasing D' at 5 V/cm, in analogy to its previously observed behavior in agarose gels. In solutions of uncrosslinked polyacrylamide, the decrease of the D' of conalbumin with polymer concentration is not significantly different from that in 2% N, N'-methylenebisacrylamide-crosslinked gels in the range of 4-14%T, while the decrease of mobility with polyacrylamide concentration is much steeper in 2% crosslinked compared to uncrosslinked polymer. Finally, delta (log D')/ delta T was found to be proportional to the retardation coefficient, KR (= -delta (log mu)/ delta T), in polyacrylamide gels. The ratio of -delta (log D')/ delta T over KR increases with field strength in the range of 5-15 V/cm.

Conalbumin↗

PubMed1995

Horizontal gel electrophoresis with sample volumes up to 1.5 mL, using a discontinuous buffer system and automated apparatus.

Horizontal gel electrophoresis has previously suffered from lack of an instrumental design that would allow for application of large sample volumes. A recently introduced commercial apparatus (HPGE-1000, LabIntelligence) remedies that problem in application to gel electrophoresis with intermittent scanning of fluorescence by using the concentrator module of that apparatus as a stacking gel reservoir. By using this technique and a 3 mL stacking gel, protein samples of up to 1.5 mL yield bands in the horizontal resolving gel that are independent of sample volume in their width, area and migration rate. A remaining procedural problem relates to the apparent nonsimultaneity of dye and protein entrance into the resolving gel, which necessitates peak characterization by absolute rather than relative mobilities.

Automation↗

PubMed1995

Capillary electrophoresis of rat liver microsomes in polymer solutions.

Rat liver microsome components were separated by capillary zone electrophoresis in buffers containing substituted agarose, agarose crosslinked polyacrylamide, polyacrylamide, polyethylene glycol and dextran of various molecular weights. The best resolution of the components was obtained with polymers of 10-21 nm geometric mean radius. Both the crude and the purified preparations of microsomes exhibit a single major peak. In a Tris-borate-EDTA (TBE) buffer, containing polyacrylamide of 5 x 10(6) molecular weight, it has a retardation coefficient, KR, of 0.77 +/- 0.02. Translation of the KR value to geometric mean radii, R, on the basis of the standard curve applicable to polymer solutions, KR vs. R, with polystyrene carboxylate size standards in both dextran and polyacrylamide solution allows one to estimate a value of R as 13-16 nm for the major microsome component. The value is smaller than expected from electron microscopic measurements (100-250 nm), possibly due to the chemical and geometric differences between microsomes and the polystyrene particles used as size standards. The crude preparation also contains a minor component which is smaller and less charged than the major component. Another component, apparently very much larger than the major one, is seen in TBE buffer but not in a potassium-N-(2-hydroxyethyl)piperazine-2'-(2-ethanesulfonic acid) buffer and is therefore thought to be an artifact of interaction with borate. After a short incubation under conditions promoting delayed microsome fusion, i.e. in presence of GTP and Mg++ and in the absence of polyethylene glycol, the electrophoretic pattern changes dramatically: it now exhibits five unretarded, highly mobile and, therefore, presumably large components in addition to the two original retarded components of the microsome and a less highly charged species similar in KR to the smaller of the original two components.

Acrylic Resins↗

PubMed1995

A comparison of resolution of DNA fragments between agarose gel and capillary zone electrophoresis in agarose solutions.

The resolving power of capillary zone electrophoresis (CZE) is compared to that of gel electrophoresis (GE) under similar conditions (agarose, similar length of DNA fragments, identical buffer) but with differences in temperature and field strength. The comparison is based on the time required to reach a desired degree of resolution by each of the two methods. A resolution parameter is developed which is equally applicable to CZE, with relatively diffuse initial conditions in the absence of stacking and measurements expressed in terms of time, and to GE, in which measurements are expressed in terms of spatial parameters. The resolution time in CZE using agarose solutions at 40 degrees C was found to be greater by at least one order of magnitude than that in GE using agarose gels. Thus, the increased migration velocity due to high field strength in CZE substantially outweighs the lower dispersion in GE.

DNA↗

PubMed1995

Band width measurement in automated gel electrophoresis apparatus: DNA dispersion in a discontinuous system and in a single buffer.

Recent commercial introduction of automated gel electrophoresis apparatus allows for band width measurements during electrophoresis and therefore promises to open up the exploitation of band width and shape for the physical characterization of charged macromolecules in the same manner in which to date quantitative gel electrophoresis had exploited electrophoretic mobility at multiple gel concentrations. The measurements demonstrate decreased band width and therefore increased resolving power for a discontinuous buffer system compared to Tris-borate EDTA buffer. The dispersion coefficients (D' = (sigma 2-sigma 2o)/t) of homogeneous DNA components appear to decrease with gel concentration when either the field strength or the DNA length is small, and increase with gel concentration when these are large. This contrasting response of D' to increasing gel concentration is presumably due to DNA stretching, which increases in proportion to DNA length and field strength, and to the progressive orientation of agarose with increasing field strength.

Autoanalysis↗

PubMed1995

Nonlinear "Ferguson curves" by two runs of the commercial automated HPGE-1000 gel electrophoresis apparatus with intermittent scanning of fluorescence.

The exploitation of gel electrophoretic migration distance to gain information of molecular and gel fiber properties depends on the functions relating mobility with gel concentration. To the degree that these are nonlinear, the definition of those functions by past methods has been excessively laborious or, in application of gel concentration gradients, based on a number of assumptions. The recent commercial introduction of gel electrophoresis apparatus capable of intermittent scanning of the pattern promised to solve these problems. The present study shows that such apparatus allows for a precise definition of a nonlinear "Ferguson curve" (mobility vs. gel concentration) in two experiments, using different gel concentrations in the eight channels of the HPGE-1000 apparatus and 5-29 scans in each during the course of an electrophoretic run. Simultaneously, these Ferguson curves are obtained for five components of a DNA ladder ranging in DNA length from 121 to 1857 bp.

Autoanalysis↗

PubMed1995

Recovery of SDS-protein and DNA using commercial automated gel electrophoresis apparatus.

The HPGE-1000 apparatus (LabIntelligence, Menlo Park, CA) is a gel electrophoresis instrument with intermittent fluorescence scanning of the migration path and with preparative capability. An electroelution cup sealed with gel is placed onto the band of interest, identified and located under computer control, and the band is electroeluted into the cup at a right angle to the orientation of the resolving gel. The correct location of the eluted band and the degree of its recovery into the elution cup are then verified on the gel pattern, visualized on the computer screen. Using that procedure, SDS-conalbumin-FLUOS was electrophoresed at 5 V/cm in a discontinuous tricinate-chloride-Tris system at loads of 0.25 to 20 micrograms, using 5% agarose (MetaPhor, FMC), 0.03% SDS gel at 5 degrees C. The horizontal gel was partitioned at the sample loading slit between a gel in Tris-tricinate (prepared at the concentrations of an operative phase ZETA) and in Tris-chloride (prepared as phase BETA). The elution cup was sealed with the latter gel and overlayered with buffer of the composition of the former. This arrangement should provide for electroelution of the band as a highly concentrated stack. At electroelution times of 2, 3.5, 4-5, 12, 15 and 15 min at 15 V/cm yields were 58, 60, 54-76, 99, 99 and 84% for loads of 0.25, 0.5, 1, 4, 10 and 20 micrograms, respectively. At the most sensitive scale of detection (13), a full-scale peak was obtained at a load of 1.7 micrograms when the fluorophore (FLUOS, Boehringer-Mannheim) to protein ratio was 10:1. Similarly, homogeneous nucleosomal DNA (146 bp), electrophoresed in 0.2 x TBE buffer at a load of 5 micrograms, was near-quantitatively recovered into the same buffer by electroelution at 15 V/cm for 2.5 or 6 min.

Autoanalysis↗

PubMed1995

A computer program for predicting recovery of SDS-protein in the automated HPGE-1000 apparatus.

The commercial automated gel electrophoresis apparatus (HPGE-1000 of LabIntelligence, Menlo Park, CA) allows one to recover the material migrating and visualized as a fluorescent-labeled band by electrophoresis into a collection cup located above the band at a right angle to the orientation of the separation path. The degree of recovery is a function of sample load (peak area), electrophoresis time at constant field strength, the mobility of the material and band width. Neglecting the latter, recovery of several SDS-proteins was measured as a function of the first three parameters. These measurements were used as a data base for a computer program capable of predicting, by interpolation of the experimental values, the time of electrophoresis needed to obtain a specified degree of recovery, or the degree of recovery obtained after a desired time of electrophoresis into the collection cup.

Autoanalysis↗

PubMed1995

Feasibility of electrophoresis of a subcellular-sized particle in polymer solutions, using automated horizontal gel apparatus.

Electrophoresis in polymer solutions of a fluorescently labeled polystyrene carboxylate particle of 46.5 nm radius was carried out in a horizontal gel electrophoresis apparatus with intermittent scanning of the migration path. Polymers of the order of 10(6) (dextran, polyvinylpyrrolidone, polyacrylamide and polyethyleneglycol) and 10(5) (hydroxyethylcellulose, polyethyleneglycol) M(r) were used. In each application, bands formed, became symmetric and narrowed with increasing polymer concentration. The decrease in dispersion coefficients, D', with polymer concentration was sharpest with the polymers of M(r) 10(6); but significant differences in the effectiveness of polymers within that group also exist which are not accounted for. Provided that the demonstrated feasibility of banding in concentrated polymer solutions will allow for separations similar to those achieved at low polymer concentrations in CZE, the method promises to overcome the inherent shortcomings of capillary electrophoresis in polymer solutions with regard to inaccessibility of bands for immunological and affinity detection as well as to preparative scale and preparative instrumental complexity.

Automation↗

PubMed1995

Molecular sieving by polymer solutions: dependence on particle and polymer size, independence of polymer entanglement.

The previous postulate of collision and displacement mechanisms of molecular sieving based on a biphasic plot of retardation coefficient vs particle radius ("R-plot") was extended and modified in four ways: i) A wider size range of particles and polymers confirmed the biphasic nature of the R-plot and, in addition, revealed a third mechanistic phase in the largest size range of particles and polymers which exhibits a positive slope in plots of retardation coefficient vs log (particle or polymer size), presumably denoting a collision mechanism. ii) Peaks of retardation in polyethyleneglycol (PEG) solutions were found with a particle M(r) of 10(7) independently of the M(r) of PEG, and with a PEG M(r) of 4 x 10(5) independently of particle M(r), showing that the retardation mechanism is not qualitatively a function of the particle/polymer size ratio as postulated previously, although quantitatively retardation is directly related to the size of particle and polymer. iii) Items i) and ii) were confirmed using band width in lieu of mobility measurement. iv) The entanglement threshold, c*, was found to decrease monotonically across the entire polymer size range in which the triphasic retardation takes place. Thus c* cannot be the sole cause for a non-monotonic change of retardation or normalized relative bandwidth with polymer size and particle size. Moreover, Ferguson plots across c* do not reflect it in any way.

Electrophoresis, Capillary↗

PubMed1994

Molecular sieving of polystyrene carboxylate of a diameter up to 10 microns in solutions of uncrosslinked polyacrylamide of M(r) 5 x 10(6) using capillary zone electrophoresis.

Capillary zone electrophoresis of polystyrene carboxylate (PSC) up to 10 microns in diameter in 0.1 to 0.9% solutions of uncrosslinked polyacrylamide (PA) of M(r) 5 x 10(6) demonstrates effective molecular sieving in that medium. The reduced mobility, however, is not constant in Tris-borate-EDTA (1x TBE) buffer containing the polymer but increases in proportion to the load of PSC and inversely to the concentration of PA unless the TBE concentration is increased 10-fold or 50 mM 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid is added to the solution or in the absence of PA. Both the Ferguson plots [log(mobility) vs PA concentration] of PSC obtained at various PSC loads and the effects of the dissociating conditions, high ionic strength and detergent, signify that the variable mobilities are those of different PSC aggregation states. Thus, only the fully associated or dissociated states of PSC provide mobilities that are constant independently of PSC load. The study shows that the information inherent in Ferguson plots regarding particle and fiber properties can be exploited for particles up to 10 microns in diameter by capillary zone electrophoresis in polymer solutions even if these particles form various aggregation states. Thus, such an analysis appears feasible in application to biologically relevant and functional complexes such as subcellular-sized particles or DNA-protein complexes, providing that these particles maintain their integrity and functionality under the selected electrophoretic conditions.

Acrylic Resins↗

PubMed1994

A computer simulation accounting for dissimilar electrophoretic behavior between two similarly curved DNA fragments due to a difference in arc-length.

Arc-shaped bent DNA fragments of the same predicted planar curvature but differing in length by 20% were compared in regard to their mobilities in 3 to 10% polyacrylamide. The longer (155 bp) fragment is retarded far more severely than the shorter (124 bp) fragment. The effect of gel concentration in promoting the retardation is far more pronounced for the 155 bp than for the 124 bp fragment. Moreover, a temperature change from 25 degrees C to 4 degrees C does not substantially affect the gel concentration dependent mobility of the 124 bp fragment while it increases the retardation of the 155 bp fragment greatly. The strong increase in retardation brought about by a mere 20% increase in the length of the arc was accounted for by a simple computer simulation of gel electrophoresis which considered the rate of passage of arc-shaped objects through a two-dimensional array of disc-shaped obstacles. Since the simulation relies exclusively on geometric factors, its success in predicting the behavior of the 124 and 155 bp DNA fragments suggests that geometric factors are largely responsible for their electrophoretic properties. The simulation can account for the strong temperature effect on the retardation of a model of the 155 bp DNA in polyacrylamide gels by showing that a decreased degree of random motion has a profound effect on the modeled 155 bp particle, but not on the modeled 124 bp DNA.

Computer Simulation↗

PubMed1994

Enhanced ethidium fluorescence of large DNA electrophoresed in gels submersed in an immiscible solvent.

Agarose gel electrophoretic separation of a lambda/HindIII DNA marker containing detectable fragments of 23 to 2 kb was carried out in the conventional submarine apparatus and in the horizontal gel slab apparatus of Wieme, using identical samples, agarose, gel width and procedure for ethidium bromide staining. In the Wieme apparatus, the gel on its microscope slide support is immersed in an immiscible solvent such as petroleum ether or silicone oil. Although band resolution and speed of migration are equivalent between gels run in the two systems, the relative fluorescence intensity of the ethidium bromide-stained bands is substantially more responsive to an increase in DNA length in the Wieme gels than in the submarine gels. The predicted relative fluorescence is by an average factor of 0.5 less than that observed after electrophoresis in the Wieme apparatus but is by an average factor of 3.4 more than that observed on bands derived from the submarine technique.

DNA↗

PubMed1993

Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis.

Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.

Animals↗

PubMed1993

The fractional volume available to prolate spheroids in a network of randomly oriented fibers obtained by computer modeling: correlation with the Ogston equation.

Computer modeling was used to measure the fractional volumes available to prolate spheroid objects in a random, inert network of fibers. The data fit the Ogston equation exactly when the object was a sphere (axial ratio = 1). When the axial ratio was increased from 1 to 9, the Ogston equation was still obeyed if the fiber concentration is multiplied by a factor, A, which increases linearly in proportion to the axial ratio. The factor A allows one to adjust the retardation coefficient derived from gel electrophoresis, KR, for spherical objects to that of prolate spheroids with axial ratios from 1 to 9. Potentially, the same adjustment of KR is possible for objects of other shapes.

Algorithms↗

PubMed1993

How far have we progressed toward automated electrophoresis in sieving media of the twenty-first century?

The automation of electrophoresis in polymeric sieving media requires (i) an objective definition of the conditions (the polymer, its concentration, solvent, buffer, pH, ionic strength, temperature) under which a particular separation proceeds most effectively; (ii) apparatus capable of zone detection, acquisition by computer, evaluation (migration distance, zone width and area) and a print-out of the number of components, their size and net charge, and the polymer conditions under which each component separates most effectively from its two neighboring zones. Both of these prerequisites of automation have been met to a first approximation at this time and, after further maturation, assembly and streamlining should be able to fill the need of the coming century for a more efficient, nonarbitrary and cost-effective mode of macromolecular and cellular particle separation. (i) The realization of the qualitative equivalence of polymer solutions and gels has greatly increased our options in the choice of sieving media. That choice can be made objectively by correlating separation efficiency (S), particle size (R) and intrinsic viscosity (eta o) of the polymer. (S) is a function of the slope, KR(R), of the Ferguson plot [log(mobility) vs. polymer concentration], or with nonlinear plots (DNA, agarose) KR(R,T). KR is at present most easily derived from transverse pore gradient gels or by conducting capillary zone electrophoresis (CZE) at multiple polymer concentrations. Pore gradient CZE appears promising. CZE also defines the free mobility unequivocally. Computer programs exist to generate KR from migration distances (times), and optimal S and polymer concentration for a particular R from KR.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoanalysis↗