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Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 55 records · Page 3Linked to original sources
PubMed1997

Protein band spreading in capillary zone electrophoresis effects of sample zone length and presence of polymer.

It is commonly accepted that intra-column zone dispersion in CZE rests on multiple mechanisms, viz. diffusion, interaction of analyte with the capillary walls, Joule heat and conductivity differences between sample zone and the surrounding buffer. The most important extra-column contributor to bandwidth is thought to be the starting zone width. The present study shows that the length of the starting zone above 10 mm is linearly related to the bandwidth of R-phycoerythrin (M(r) 290.10(3)). Below that length, bandwidth demonstrates a plateau preceded by a slight rise. Within the 'plateau range', the ratio of bandwidth to effective capillary length is close to constant while it is independent of electric field strength in the range of 37 to 370 V cm-1 and of protein concentration in the range of 0.1 to 1000 micrograms ml-1. The experimental observations support the notion that the analyte-wall interaction is the determining source of intra-column zone dispersion. A slight rise observed at initial zone lengths of less than 2 mm was accounted for by a diffusion model taking into account a non-local initial concentration of analyte. The presence of polyethyleneglycol in the buffer within a concentration range up to 6% does not affect bandwidth. Above that concentration, the level of constant bandwidth is raised.

Electrophoresis, Capillary↗
PubMed1997

Preparative electrophoresis in a concentrated polymer solution: automated procedure for microsome isolation.

A recently introduced automated gel electrophoresis apparatus with an intermittent scanning of the migration path, the HPGE 1000 apparatus (LabIntelligence, Belmont, CA) has a unique preparative feature: A recovery cup can be automatically positioned on the band of interest, which is then electroeluted with fluorescence monitoring of recovery. Electroelution time is extended stepwise until quantitative recovery is attained. This preparative procedure has been extended from electrophoresis in gels to that of subcellularsized particles in concentrated polymer solutions Chang et al., Electrophoresis 17, 776-780, 1996). However, that application required manual rather than computer-directed positioning of the recovery chamber over the band. The present report details a modification of instrumentation and procedure by which automated operation was restored. The modified procedure extends the benefits of automation and known quantitative recovery to isolation of particles too large to enter into gels.

Animals↗
PubMed1997

Electrophoresis of proteins in semidilute polyethylene glycol solutions: mechanism of retardation.

The retardation of proteins in the M(r) range of 15-500 kDa in capillary electrophoresis conducted in semidilute solutions of the polymer polyethylene glycol (M(r) range 0.2-8.0 X 10(6)), was measured. The purpose was to test the predictions of the scaling theory with regard to the relation of retardation to (a) the M(r) of the polymer, (b) the concentration of the polymer, and (c) the radius of the protein particles. These predictions derive from a mechanism that relates retardation to the screening length of the polymer solution, viewed as the average distance between the entanglement points of polymer chains. For the molecular weight range from 60 to 500 kDa of (near) spherical proteins, the retardation was found to be related to polymer concentration c as mu/mu(0) = exp(-Ac0.69) where mu/mu(0) is the retardation expressed as the ratio between the mobility in polymer solution and that in free solution. The value of the exponent of 0.69 is in close agreement with the value of 0.75 predicted by the scaling theory. Parameter A was found (a) to scale as the 0.04th power of M(r) (polymer), approximating the predicted value of 0; and (b) to be proportional to particle radius as predicted. All measured values of retardation were independent of electric field strength in the range of 37-370 V/cm. Thus, experimental findings are consistent with the mechanism relating electrophoretic retardation to the screening length of the polymer network in the specified molecular weight range of proteins. Under the same conditions, log(mu/mu(0)) of proteins with M(r)'s less than 60 kDa (a) scales as the -0.06th power of M(r) (polymer), and (b) is proportional to polymer concentration, suggesting a retardation mechanism that is not related to the screening length.

Capillary Action↗
PubMed1997

Isolation of subcellular-sized particles separated by electrophoresis in dilute polymer solution, using commercial electrophoresis apparatus with intermittent scanning of fluorescence.

Resolution of subcellular-sized particles in electrophoresis employing semi-dilute polymer solutions as "sieving media" improves as the polymer concentration is decreased. Therefore, the previously reported conditions of preparative electrophoresis of microsomes, using concentrated (12%) polyvinylpyrrolidone (PVP) solutions, while solving the problem of non-entrance of large particles into "sieving media", do not provide adequate resolving capacity, as exemplified by failure of the microsome preparation used, to resolve in the manner of gels or dilute solutions. The present report provides the conditions under which the HPGE-1000 apparatus can be preparatively applied when the electrophoretic separation is effectively conducted in a dilute polymer solution. The isolation of three microsome components under those conditions constitutes the first application of "particle sieving", i.e., a separation due preponderantly to size and shape differences, at a preparative scale.

Animals↗
PubMed1997

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.

Conalbumin↗
PubMed1997

Improved sensitivity of detection with the commercial automated gel electrophoresis (HPGE-1000) apparatus through modification of its optical system.

In a representative application to a fluorescently detectable protein of commercial automated gel electrophoresis apparatus (HPGE-1000, LabIntelligence, Belmont, CA) the sensitivity of detection by fluorescence was significantly increased by elimination of the mirror below the gel tray. That increase in detection sensitivity is due to a decrease of fluorescent background noise by nearly one order of magnitude, overcompensating a decrease in signal by a factor of two. The resulting increase in signal/noise ratio, i.e., detection sensitivity, should allow for lowered sample loads by which the band width is reduced with benefits to resolution.

Autoanalysis↗
PubMed1996

Improved resolution in the gel electrophoresis of proteins by a periodically interrupted electric field.

The capability of the commercial gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE-1000, LabIntelligence) to provide time-dependent zone dispersion allows one to quantitate resolution. Using a model protein separation, that between phycoerythrin and fluorescein-labeled conalbumin, resolution was compared between separations conducted at a constant field strength of 80 V/cm and one conducted in 10-s pulses of the same field strength, interrupted periodically by 120 s in the absence of an electric field. Resolution was improved by a factor of two in the discontinuous application of the electric field compared to that obtained in its continuous application. Similarly, the intermittent application of 80 V/cm for 10 s, followed by 120-s pauses, gave rise to twice the resolution obtained from a continuous application of 7 V/cm.

Computer Graphics↗
PubMed1996

Application of the commercial gel electrophoresis apparatus with intermittent fluorescence scanning to a nonfluorescing protein.

Gel electrophoretic instrumentation has taken a quantum jump forward with the commercial introduction of an apparatus which, after loading of the sample and initiation of electrophoresis, provides real-time gel patterns at desired time intervals, with a computer printout of mobility values characterizing each band and the means to isolate each desired band with known and maximizeable recovery. However, a major limitation of that apparatus has been that it employs fluorescence detection and therefore requires the fluorescent labeling of the macromolecules of interest. That limitation was first overcome by E. Gombocz and E. Cortez (Application Note 8, 1994, LabIntelligence, Belmont, CA) in the detection of nonfluorescing carrier ampholytes. In that application, fluorescent, immobile (uncharged) umbelliferone was added to the gel to provide a uniform background of fluorescence upon excitation at 280-360 nm. The isoelectric carrier ampholyte zones could be detected as inverted peaks due to their reduction of the fluorescence intensity of umbelliferone. A similar approach was applied to a representative SDS-protein, conalbumin-SDS, in the present study, replacing umbelliferone in the gel by a fluorescing paper sheet in contact with the lower external surface of the electrophoresis cell. Passage of the proteins reduced the intensity of the light excitation incident on the fluorescent paper so as to decrease the emitted fluorescence signal and allow for the detection of the proteins as "inverted peaks." Presumably, the reduction of background fluorescence is due to the absorbance at 280 nm of the protein passing through the gel, and reduction of the incident light intensity by that absorbance. The resulting detection of the representative unlabeled SDS-protein by "fluorescence reduction" was found to be less sensitive by a factor of 10-20 than detection of the fluorescently labeled protein (at a molar ratio of fluorescein carboxylate to conalbumin of 1/1). The area of the inverted bands of conalbumin-SDS was found to be independent of migration distance.

Conalbumin↗
PubMed1996

Application of gels of 0.5 mm thickness to electrophoresis in the automated HPGE-1000 apparatus: improved resolution.

Gel electrophoresis in commercial automated apparatus (HPGE-1000, LabIntelligence, Menlo Park, CA) is conventially conducted in gels of 3 mm thickness at about 15 V/cm. Since the intermittent scanning of the gel allows one to measure band width as a function of migration time, resolution may be evaluated quantitatively. Comparing the value of resolution between two proteins in electrophoresis on agarose gels of 0.5 and 3.0 mm thickness and at various field strengths, it was found that within the given Joule heat dissipation capacity of the apparatus, resolution between the proteins is improved when the gel thickness is reduced from 3.0 to 0.5 mm, which allows for an increase in field strength from 15 V/cm, conventional for that apparatus, to 45 V/cm.

Autoanalysis↗
PubMed1996

Reproducibility of mobility in gel electrophoresis.

The quantitative exploitation of gel electrophoresis to yield molecular and gel fiber properties rests on the assumption that mobility is characteristic of the macromolecule migrating as a band and is a physical constant for any system defined by pH, ionic strength and temperature. This assumption has not been tested intra-experimentally in previous literature. With the commercial introduction of automated gel electrophoresis apparatus, the collection of multiple mobility data during a single run without additional expense of labor has made it possible to test the assumption. As a start, we undertook that test for three proteins and their sodium dodecyl sulfate (SDS) derivatives, in agarose and polyacrylamide gel electrophoresis, various field strengths, continuous and discontinuous buffers, as well as intra- and interexperimentally. It was found that in agarose gel electrophoresis conducted in a single buffer, the standard deviation of mobility over a wide concentration range ranges intra-experimentally from 0.2 to 1.3% for two globular proteins and 1.4 to 5.3% for the same proteins derivatized with SDS. Interexperimentally, it was 3% in the single case tested to date. The standard deviation in polyacrylamide appears to be higher, varies in inverse relation to the mobility value, i.e. increases with gel concentration in the range of 11 to 19%T, and varies substantially between the two SDS-proteins investigated. Mobility in a discontinuous buffer system decreases continuously due to the decreasing leading phase/trailing phase ratio along the migration path. The decrease is sharpest in the "nonrestrictive" stacking gel.

Autoanalysis↗
PubMed1996

Enhanced field strength and resolution in gel electrophoresis upon substitution of buffer by histidine at its isoelectric point.

Gel electrophoresis in isoelectric buffers, recently introduced by R. Westermeier and H. Schickle (Electrophoresis '95, Paris, Abstract No.3, 1995), was applied to the automated HPGE-1000 apparatus in the expectation to be able to increase the field strength under the limiting conditions of heat dissipation capacity and voltage of that apparatus. A previous attempt to achieve that aim by reduction of gel thickness had not yielded more than a twofold increment in resolving power. Replacing 0.2 X Tris-boric acid-EDTA (TBE) buffer, conventionally applied in the apparatus at 15 V/cm, by 0.05 M histidine, pH 7.6 (close to the pI of 7.47), allows one to increase the field strength to 60 V/cm, thus providing a nearly fivefold increment in resolution under otherwise identical conditions (fluorescein carboxylate-labeled conalbumin-sodium dodecyl sulfate (SDS) and soybean trypsin inhibitor-SDS samples, 10 degrees C, 4% MetaPhor agarose). An additional decrease in band dispersion can be obtained by decreasing the starting zone width through buffer dilution in the sample phase.

Boric Acids↗
PubMed1996

Separation and isolation of subcellular-sized particles by electrophoresis in polymer solution using the commercial scanning apparatus.

Electrophoresis of fluorescently labeled rat liver microsomes and polystyrene carboxylates of 10 and 30 nm radius was conducted in buffered 10-15% polyvinylpyrrolidone (M(r) = 10(6) solutions, using a horizontal gel electrophoresis apparatus with intermittent scanning of fluorescence (HPGE-1000, LabIntelligence). Banding, constant migration rates and Ferguson plots were obtained in these polymer solutions. The major microsome band detected by the automated scan was located visually on the gel by means of its fluorescein label and was isolated by volumetric withdrawal, recovery was monitored by scanning and ascertained to be near quantitative after three consecutive steps, in each of which 30 microL were withdrawn. This preparative method promises to be generally applicable to particles that are too large to enter into gels.

Animals↗
PubMed1996

Mechanisms of retardation of rigid spherical particles with 3 to 1,085 nm radius in capillary electrophoresis, using buffered polyacrylamide (molecular weight 5 x 10(6)) solutions.

Subjecting particles in the size range of 3 to 1085 nm radius (R) to capillary electrophoresis in buffered solution of entangled uncrosslinked polyacrylamide (M(r) 5 x 10(6)), it was found that particle size-dependent retardation ("molecular sieving") becomes electric field- and particle size range-dependent once the particle size exceeds 15-20 nm in radius. The field strength dependence of the retardation coefficient [KR = d(log mobility)/ d(polymer concentration] and the positive or negative sign of dKR/dR suggest the existence of two different mechanisms of molecular sieving depending on the particle size range: particles with diameters less than the screening length (or blob size) of the polymer network are thought to penetrate into the available spaces within a discontinuous polymer network; particles with diameters larger than the screening length (or blob size) of the polymer network are thought to undergo size-dependent retardation by exerting shear stress against polymer chains, and displacing them, so as to cause local deformations in a continuous polymer network. A limit in the separating capacity of molecular sieving, due to a sharp increase in the rate of band widening with polymer concentration, was found when the value of the retardation coefficient exceeded 60 (mL/g).

Acrylic Resins↗
PubMed1996

Interpretation of electrophoretic band shapes by a partition chromatographic model.

Measurements of the shape of electrophoretic bands of phycoerythrin and conalbumin have been made at regular intervals during migration in agarose gels. Analysis of the peak shapes suggests the existence of a significant degree of asymmetry. This is to be contrasted with the symmetry around the peak associated with the generally assumed Gaussian band. The degree of asymmetry of the bands decreased as a function of time and increased with agarose concentration. A similar experiment on DNA indicated constancy of the degree of asymmetry as a function of time. These results can be interpreted as, but do not prove the validity of, a nonlocal diffusion equation which generalizes a theory originally put forth by Giddings and Eyring (J. Am. Chem. Soc. 1955, 59, 416-420). The results may be significant in framing a measure of the resolvability of electrophoretic peaks.

Electrophoresis, Agar Gel↗
PubMed1996

The relative contributions of dispersion and diffusion to band spreading (resolution) in gel electrophoresis.

DNA of approximately 2 kbp in length was previously found not to diffuse significantly in 1-1.5% agarose gels in the absence of an electric field, but to disperse during electrophoresis (Yarmola, E., Chrambach, A., Electrophoresis 1995, 16, 345-349). Accordingly, a process distinct from diffusion, and responsible for band spreading with migration time in gel electrophoresis, was defined as dispersion. Correspondingly, the diffusion coefficient, D(diff), was distinguished from a dispersion coefficient, D(disp). For DNA of approximately 1, 2 and 3 kbp, D(diff) and D(disp) were measured in agarose gel electrophoresis (1.0% SeaKem GTG). In that order of DNA length, D(disp)/D(diff) was found to increase from 5 to 15 to 45, showing that with increasing DNA length, time-dependent band spreading, and thus resolution in gel electrophoresis, is governed predominantly by dispersion, not diffusion. It is assumed that the essential part of electrophoretic dispersion is due to entanglement of the DNA molecule in the gel. Indirect evidence for such an entanglement derives from the observation of peak asymmetry and its interpretation by the Giddings-Weiss model.

DNA↗
PubMed1995

Determination of optimally resolving gel concentration and migration time (path) in gel electrophoresis.

The notion of a mathematically defined optimally resolving gel concentration for components of a pair of molecular species of any given size was developed by Rodbard et al. (Electrophoresis and Isoelectric Focusing on Polyacrylamide Gel, pp. 28-62, de Gruyter, Berlin, 1974) 21 years ago. The mathematical treatment was incorporated into a computer program (T-OPT) for mainframe computers which upon input of the slope and intercept on the mobility axis of the Ferguson plots of the two components, electrophoresis time and temperature, yielded plots of resolution vs gel concentration. The same algorithms were later incorporated into the program ELPHOFIT for personal computers. Ideality of diffusion spreading and zero initial zone width were assumed along with a Gaussian peak distribution and an equal area for both components. Moreover, these programs failed to respond to the practical question of the migration time (or path) required for the resolution at the optimal gel concentration, although an independent program predicting the course of resolution under the assumption of free diffusion band spreading in gels (DAR-001) had been devised by Rodbard for application in preparative elution-PAGE. The present work advances the technology for predicting resolving conditions by presenting a computer program which allows the user (i) to predict the gel concentration which is optimal for obtaining the desired degree of resolution at any migration time, (ii) to prescribe the minimal degree of resolution between two band distributions one wishes to achieve, and (iii) to predict the migration time (or path) required at the optimal gel concentration for the resolution of the two components. The program is written in MATLAB and can be used by any computer that supports MATLAB language.

Algorithms↗
PubMed1995

Fluorescent labeling of DNA with ethidium homodimer without measurable decrease in DNA mobility: application to automated gel electrophoresis apparatus.

Mobilities of DNA, fluorescently labeled with ethidium homodimer (EtD), and normalized to the mobility of the 50-bp fragment [Rf(50)], are constant with time of electrophoresis, permitting one to conduct studies on DNA mobility in an automated electrophoresis apparatus with fluorescence detection. DNA mobility decreases with an increasing binding density of ethidium homodimer, as previously reported by others and expected since the dye decreases both the net charge and the conformational flexibility of the DNA. However, it was found that this effect of ethidium binding on electrophoretic mobility becomes undetectable at binding densities less than 1 EtD/40 bp. This implies that for the purposes of electrophoretic analysis in an apparatus with fluorescence detector, DNA with ethidium homodimer intercalated at dye-DNA ratios less than 1/40 bp behaves like unlabeled DNA and may be assumed to be in a conformation similar to that of the unliganded DNA. Necessarily, the low labeling ratio lowers the sensitivity of detection and, in particular, increases the load requirement for a full-scale band height required for the analysis of bandwidth and band shape during electrophoresis.

DNA↗