Biomedical subjects

A Chrambach

Publications and source records attributed to A Chrambach.

At least 37 records · Page 2Linked to original sources

PubMed1999

Capillary zone electrophoresis of rigid submicron-sized particles in polyacrylamide. Solution selectivity, peak spreading and resolution.

Submicron-sized rigid particles can be separated in a size-dependent fashion by electrophoresis in free solution. Yet it has remained unknown whether the presence of polymers in the solution confers an advantage in size-dependent separation of submicron particles and their resolution. The present study addresses that question, using capillary zone electrophoresis of carboxylate modified polystyrene latex microspheres of 55, 140 and 215 nm radius in solutions of linear polyacrylamide in the M(r) range of 0.4.10(6) to 1.14.10(6). Selectivity of particle separation increases in direct relation to the polymer concentration in the concentration range of 0 to 1% (w/v). Selectivity was found to increase with M(r) of the polymer for the particle sets of 55/140 (nm/nm) and 140/215 (nm/nm) but to decrease with polymer M(r) for the 55/215 (nm/nm) set. Peak spreading is a complex and, in the case of the largest particle, non-monotonic function of polymer concentration, with a minimum at concentrations around the entanglement threshold, c*. Consequently, resolution of the 55/215 and 140/215 (nm/nm) sets also exhibits a maximum around the entanglement threshold while resolution for the 55/140 (nm/nm) set increases with a rise of polymer concentrations above c*. Within the range of optimally resolving polymer concentrations there also occurs a maximum of resolution for all particle sets at a field strength in the range of 150 to 250 V cm-1.

Acrylic Resins↗

PubMed1999

Capillary electrophoresis of subcellular-sized particles.

Utilization of capillary electrophoresis (CE) for characterization and analytical separation of submicron- and micron-sized organic and inorganic particles as well as biological vesicles is reviewed. CE has been applied to charged polystyrene size standards, inorganic and organic colloidal particles, lipoprotein particles, liposomes, microsomes and viruses. These particle separations generally occur in a size-dependent manner and provide values of electrophoretic mobility which are in good agreement with those obtained by other electrophoretic techniques.

Electrophoresis, Capillary↗

PubMed1999

Mass spectrometric analysis of the electroeluates of fluorescent proteins after preparative electrophoresis in the automated HPGE-1000 apparatus.

Bands of green fluorescent protein (GFP) and R-phycoerythrin (PHYCO) in gel electrophoresis on the automated apparatus for gel electrophoresis with periodic fluorescence scanning (HPGE), the HPGE-1000 apparatus, were retrieved from the gel by electroelution. While PHYCO was recovered in a single volume of electroeluate buffer after the predicted migration time, GFP fluorescence was lost under the same conditions and could only be recovered using multiple changes of electroeluate buffer. The multiple volumes of buffer necessitated pooling, concentration, and storage, conditions under which a minor GFP component, GFP-II, formed artifactually. PHYCO after electroelution also exhibits a minor component present in the original preparation. The electroeluate of GFP, transferred into a mass spectrometer after pooling, concentration and storage, is indistinguishable in mass from the original preparation.

Automation↗

PubMed1999

PhastSystem electrophoresis in beta-octylglucoside containing gels with immunodetection of a nondenatured vesicle-associated membrane protein.

Recombinant vesicle-associated membrane protein (rVAMP), implicated as a participant in membrane exocytosis and fusion (a "SNARE protein"), was subjected to gel electrophoresis in the miniaturized gels of the PhastSystem (Pharmacia) containing the nondenaturing, nonionic detergent beta-octylglucoside (OG), followed by immunodetection of the protein. Three major components of nondenatured rVAMP are detected by Western blotting both in 0.5% OG and in the absence of detergent. Their separation increases with increasing gel concentration above 7%T. Ferguson plot analysis indicates that the three species of VAMP are size isomers (i.e., they differ in size but share a common surface net charge density), the common point of intersection of the plots (mu-point) being a measure of their common free mobility. By the criteria of size and free mobility (related to surface net charge), VAMP components I, II and III in 0.5% OG-containing buffer are indistinguishable from components II, III and IV, respectively, observed in the absence of the detergent. The feasibility of immunodetection of nondenatured rVAMP on gels containing nondenaturing detergents opens up the possibility of gaining biochemical information regarding nondenatured SNARE protein complexes and SNARE proteins linked to membrane fragments.

Animals↗

PubMed1999

Gel electrophoretic distinction between toxic and nontoxic forms of beta-amyloid (1-40).

The in vitro toxicity of synthetic beta-amyloid (1-40) correlates with its binding to Congo red (CR). Potentially, therefore, CR binding to the beta-amyloid containing neuritic plaques in Alzheimer's disease could be used diagnostically. Using polyacrylamide under nondenaturing conditions, the present study shows that both CR binding and nonbinding synthetic beta-amyloid exhibits multiple charge-isomeric and size-isomeric species. The CR binding species exhibit values of free electrophoretic mobility, related to the surface charge density of the protein, which are less than those of the CR non-binding species within 95% confidence limits. Since surface net charge and solubility are correlated, the decreased solubility of the CR binding species may be responsible for the relative abundance and CR binding of beta-amyloid in the neuritic plaques of Alzheimer patients.

Alzheimer Disease↗

PubMed1999

Capillary zone electrophoresis of proteins in semidilute polymer solutions: inter- and intra-polymer predictability of size-dependent retardation.

The retardation of three "spherical" proteins with Stokes' radii of 2.0, 2.4, and 3.0 nm (35-104 kDa) was studied in capillary zone electrophoresis (CZE), using semidilute solutions of polyethylene glycol (PEG), linear polyacrylamide (PA), and polyvinyl alcohol (PVA). The purpose was to test the models predicting that the ratio of particle radius, R, to the mesh size of polymer network (the correlation or screening length of a semidilute polymer solution), xi, directly governs the size-dependent retardation in the form: mu/muo = exp (-R/xi). Here xi = kc-0.75, where c is polymer concentration and the numerical factor kcan be calculated based on polymer molecular weight. In application to polymers in a "good solvent" (PA and PEG in the aqueous buffer) and to proteins of 2.4 and 3.0 nm radius, that relation between relative mobility and R/xi was found to be obeyed for PA, while for PEG the value of k derived from retardation experiments significantly exceeded that which was theoretically calculated. Thus, the retardation appears to be polymer-specific, rather than universal, even for polymers in a "good solvent". It is suggested that, in that case, retardation of proteins of R > 2 nm be quantitatively described in the form mu/muo = exp[-p(R/xi], where p is a parameter depending on monomer type and/or polymer polydispersity. For PVA, the logarithm of mu/muo was found to be linearly related to c (in line with the prediction that the aqueous buffer is a "poor solvent" for this polymer) and to be near-independent of R.

Animals↗

PubMed1998

Asymmetry of protein peaks in capillary zone electrophoresis: effect of starting zone length and presence of polymer.

The asymmetry of R-phycoerythrin (M(r) = 240,000) peaks in capillary zone electrophoresis measured as In[(tm-t1)/(t2-tm)], where tm, t1 and t2 are migration times of the peak mode and at the intersection of the peak width at half-height with the ascending and descending limbs, respectively, was found to undergo a transition from negative to positive values with increasing starting zone length. The transition is compatible with a mathematical model of peak dispersion which assumes that an interaction of protein with the capillary walls governs the evolution of the peak during capillary zone electrophoresis. Models assuming a final peak shape defined solely by longitudinal diffusion, or by a heterogeneity with regard to mobility or by a conductivity difference between analyte zone and background electrolyte, have failed to give rise to a change in the sign of peak asymmetry when the starting zone length is varied. The presence of polyethylene glycol in the buffer within a concentration range up to 4% does not appreciably affect the peak asymmetry regardless of whether the concentration regime is dilute or semi-dilute. Above 4% of polyethylene glycol, the asymmetry becomes nearly independent of starting zone length, and progressively negative with increasing polymer concentration. The concentration range at which the transition from negative to positive asymmetry disappears coincides with that at which the average mesh size of the polymer network falls below the size of the protein.

Electrophoresis, Capillary↗

PubMed1998

Reconstitution of calcium-triggered membrane fusion using "reserve" granules.

Calcium-gated secretion of proteins involves the transfer of "reserve" granules, exocytotic vesicles that are cytoplasmic and, hence, plasma membrane-naive, from the cell interior to the surface membrane where they dock prior to fusion. Docking and subsequent priming steps are thought to require cytoplasmic factors. These steps are believed to induce fusion competence. We have tested this hypothesis by isolating reserve granules from sea urchin eggs and determining under which conditions these granules will fuse. We find that isolated reserve granules, lacking soluble cofactors, support calcium-dependent membrane fusion in vitro. Preincubation with adenosine 5'-3-O-(thio)triphosphate and guanosine 5'-3-O-(thio)triphosphate did not prevent fusion. Thus, isolated reserve granules have all the necessary components required for calcium-gated fusion prior to docking.

Adenosine Triphosphate↗

PubMed1998

Enhanced detection sensitivity of "fluorescence reduction" by shifting the analyte absorbance spectrum and use of a fluorescent paper with higher signal/noise ratio.

Nonfluorescing protein bands can be detected by the fluorescence optics of the commercial gel electrophoresis apparatus with automated scanning of the migration path (HPGE-1000, LabIntelligence, Belmont CA), taking advantage of the decrease of emission from a fluorescent paper placed below the gel by the absorbance of proteins ("fluorescence reduction"). That decrease of fluorescence gives rise to an inverted protein peak. Nonfluorescent colorless proteins appear to reduce the intensity of light emitted from the fluorescent paper due to absorbance of incident and emitted light. When the absorbance spectrum only slightly overlaps with the excitation and emission spectra of the fluorescent paper, that reduction is weak, and detection sensitivity in that application is consequently only 1/30 of that of fluorescent proteins. By contrast, when the protein is colored so that its absorbance spectrum overlaps widely with the excitation and emission spectra of the fluorescent paper, the sensitivity of "fluorescence reduction" equals 1/4 to 1/5 of that obtained for fluorescent proteins. Bands detected by "fluorescence reduction" provide a quantitative measure of protein load and mobility. The area of the inverted bands is proportional to protein loads up to 16 microg/lane of the gel tray. A theory of "fluorescence reduction" is presented which accounts for the existence of a linear relationship between band area and load.

Animals↗

PubMed1998

Separation and microgram-scale isolation of sea urchin egg granules by electrophoresis in polyvinylpyrrolidone solution, using horizontal gel electrophoresis apparatus with fluorescence detector.

A homogenate of sea urchin (Lytechinus pictus) eggs rich in exocytotic membrane vesicles (granules) was subjected to analytical and preparative electrophoresis in the commercial automated horizontal gel electrophoresis apparatus (HPGE-1000, LabIntelligence, Belmont, CA) capable of intermittent scanning of the migration path, using buffered solutions of polyvinylpyrrolidone (PVP). The nonfluorescent granules were detected by "fluorescence reduction", i.e., a decrease of fluorescence intensity due to the absorbance and/or light scattering properties of the particle. Granules migrated at linear migration rates in buffers ranging from 0 to 2.5% PVP. Two bands were observed and optimally separated in 1.5% PVP solution. As shown by sodium dodecyl sulfate (SDS)-polypeptide patterns, the material recovered from the bands was qualitatively indistinguishable from the two major fractions A and C of granules previously separated by free-flow electrophoresis in the absence of polymer. Ferguson plot analysis failed to provide the sizes of the granules in view of the narrow PVP concentration range available for mobility measurement and the unavailability of chemically homogeneous size standards.

Animals↗

PubMed1998

Separation of viable from radiation-induced apoptotic lymphocytes by free-flow electrophoresis.

A human lymphocyte population undergoing apoptosis in vitro due to gamma-irradiation was fractionated by free-flow electrophoresis in triethanolamine--Na-acetate buffers, containing up to 50 mM NaCl, with pH 6.0, 7.2 and 8.5, made isotonic by addition of sucrose. As shown by a flow cytometric analysis of the eluate, the distribution of apoptotic lymphocytes is shifted to the range of higher electrophoretic mobilities relative to that of viable ones at pH 8.5, yielding cell fractions enriched in apoptotic cells by a factor of 3 to 5. The difference in rates of electrophoretic migration observed at a mildly alkaline pH but not at a neutral or mildly acidic one suggests that the surface of apoptotic lymphocytes is more acidic than that of viable ones.

Apoptosis↗

PubMed1998

Preparative electrophoresis in "sieving media" of subcellular-sized particles.

The commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE-1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11-15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE-1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by "fluorescence reduction". The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr = 10(6)) solution in 25-50% yield (0.24-4 microg/8 lanes of the HPGE-1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients, KR = d(log mobility)/d(polymer concentration), are not ascertained.

Animals↗

PubMed1998

Towards predicting mobility and resolution in polymeric media: some first steps.

Particle size-dependent retardation ("molecular sieving") in electrophoresis can be achieved in polymer solutions or gels. In the semidilute concentration range of polymer solutions, mobility of "rigid, spherical" particles can be predicted from their size (in the size range less than 20 nm radius), the screening length specific for the particular polymer and a constant that can be experimentally determined for a polymer. That constant could be universal for all hydrophilic uncharged polymers. Band spreading in polymer solutions is constant over a range of particle sizes and polymer concentrations and increases beyond that range. Presumably, the critical division between the two ranges occurs when the diameter of the particle exceeds the screening length of the polymer network. Resolution, defined as separation divided by the sum of bandwidths, can thus be predicted for a limited particle size and polymer concentration range. In gels, resolution can be estimated from the slopes and free mobility intercepts of the Ferguson plot. However, the previous computation of resolution needs to be revised in view of the fact that band spreading in gels proceeds in proportion to time, presumably through interaction with the polymer, and not as a function of the square root of time as would be the case if band spreading resulted from diffusion.

Diffusion↗

PubMed1998

Parallelism between width and asymmetry of peaks of rigid, spherical particles in capillary zone electrophoresis using polymer solutions.

Peak width and peak asymmetry of rigid spherical particles in the size range of 3-100 nm radius (R) were measured in capillary zone electrophoresis (CZE), using buffered uncross-linked polyacrylamide of Mr 5.0 X 10(6). Polymer concentration-dependent spreading of peak width and peak asymmetry were found to parallel one another. The parallelism holds whether the particle size is within the "small" (R < 20 nm) or "large" (R > 20 nm) size ranges previously found to differ in the mechanism of particle size dependent retardation of electrophoretic migration (S. P. Radko and A. Chrambach, Electrophoresis 1996, 17, 1094-1102). In application to the "small" particle size range, the parallelism between band width and band asymmetry can be qualitatively interpreted to be consistent with the Giddings-Weiss mechanism (G. H. Weiss et al., Electrophoresis 1996, 17, 1325-1332) of electrophoresis in polymer-containing media which postulates a dependence of band width and band asymmetry on the equilibrium between "stationary" and "mobile" states of the particle.

Acrylic Resins↗

PubMed1998

The band areas of proteins determined by fluorescent scanning in the commercial automated gel electrophoresis apparatus.

An automated gel electrophoresis apparatus, recently available commercially, allows one to follow the band during electrophoresis in real time, and lends itself therefore to an evaluation of bandwidth as a function of migration time (the dispersion coefficient), resolution and band shape. These determinations assume the constancy of band area with migration time and at various gel concentrations. The purpose of the present study was to verify these assumptions. Representative proteins and sodium dodecyl sulfate (SDS)-proteins, either natively fluorescent or fluorescein carboxylate labeled, were found to exhibit band areas which approach constancy as a function of migration time in both agarose and polyacrylamide gel electrophoresis, provided that (i) the protein concentration under the band was low enough to obviate self-quenching of fluorescence; (ii) the separation of the protein of interest from contaminants had progressed sufficiently during the time at which band areas were measured; (iii) the baseline under the peak was sufficiently well defined. However, band areas decrease with increasing gel concentration. Protein peaks exhibited leading and trailing tails. The ratio of the combined tail area to total area appeared to be near-constant at varying migration times. However, that ratio increases with increasing gel concentration. The tail area does not appear to be an artifact of fluorometric detection since it is reproduced upon fluorimetric analysis of the protein eluted from gel slices after electrophoresis. However, it may be due to photochemical destruction under the conditions of repetitive fluorometric peak detection.

Adsorption↗

PubMed1998

Mechanistic insights derived from retardation and peak broadening of particles up to 200 nm in diameter in electrophoresis in semidilute polyacrylamide solutions.

Rigid spherical particles in the size range of 5-200 nm diameter were subjected to capillary zone electrophoresis (CZE) in semidilute solutions of uncross-linked polyacrylamide of M(r) 5, 7 and 18 x 10(6) (PA-5, -7 and -18, respectively) of varying concentrations up to 1.6% and at field strengths varying from 68 to 270 V/cm. For all particles under study, the experimental Ferguson plots, log(mobility) vs. polymer concentration, permit a linear approximation. Their slope, the retardation coefficient KR = delta log (mobility)/delta (concentration), for particles smaller than 30 nm in diameter increased with particle size in PA-5 and -7 independently of electric field strength and polymer M(r). The KR of particles of 30 nm in diameter or more was found to be independent of particle size at the lowest field strength used but to decrease with it at the higher values of field strength. The decrease was parallel but shifted to higher values of retardation when the polymer M(r) increased from 5 to 7 x 10(6). With a decreasing ratio of average mesh size of the polymer network, zeta, to particle radius, R, the approach to "continuity" of the polymeric medium (zeta/R << 1) with both increasing particle size and polymer concentration does not result in the retardation behavior expected according to the macroscopic (bulk) viscosity of the solution. These experimental observations were hypothetically interpreted in terms of a transition to a retardation mechanism comprising the formation of a polymer depletion layer near the particle surface--polymer solution interface. Peak width exhibited an overall increase with PA-7 concentration for all particles studied. For particles of 30 nm in diameter or less, the increase was steepest when the radius of the particle was approximately commensurate with zeta at a given polymer concentration. For the largest particle, 205 nm in diameter, peak broadening with polymer concentration was found to correlate linearly with peak asymmetry. CZE of the particles in PA-18 solutions revealed abnormal behavior, with both mobility and peak width remaining near-constant up to a concentration of 0.08% and sharply declining at higher concentrations. The decline of relative mobility is the same-for the entire particle size range used, while peak width declines in direct relation to particle size.

Acrylic Resins↗

PubMed1998

Preparative application of commercial automated gel electrophoresis apparatus to subcellular-sized particles: sequential isolations, fractions re-run, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, yield and purity.

The analytical and preparative potential of automated gel electrophoresis apparatus with intermittent fluorescence scanning of the migration path, the HPGE-1000 apparatus (LabIntelligence, Belmont, CA) was further developed in application to subcellular-sized particles. Resolution between two rat liver microsome components in agarose (MetaPhor) gel electrophoresis was found to increase with decreasing agarose concentration to 0.04%. It was less, even in an agarose solution at that low concentration, than that in laterally aggregated 4% polyacrylamide gel. The three components of the microsomal preparation were sequentially isolated from 0.6 and 0.8% agarose gel electropherograms. One fraction when re-electrophoresed was found to exhibit the original mobility and did not give rise to the other components. Yields of each component were near-quantitative after one or two electroelution steps. Based on protein content, no impurities could be detected in two of the microsome fractions; the third fraction contained 2% of nonmicrosome impurity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of all three microsome fractions were indistinguishable from one another and from that of the unfractionated microsome preparation.

Animals↗

PubMed1997

Capillary electrophoresis of DNA fragments in 9 to 20% uncrosslinked polyacrylamide gels: unique separating capacity hypothetically related to maintenance of random-coil DNA conformation independently of gel concentration.

DNA fragments (0.1 to 2 kb) were separated by capillary electrophoresis (CE) in 9 to 20% uncrosslinked polyacrylamide gels with a resolving power ranging from 3 to o.1 million theoretical plates/meter across that DNA size range. The unique feature of electrophoresis in 18 to 20% uncrosslinked polyacrylamide is that it provides a method capable of resolving charge isomeric species of DNA fragments (0.4 to 2 kb), confirming a previous report by Heiger et al. [Heiger DN, Cohen AS, Karger BL. J Chromatogr 516 (1990) 33-48]. A similarly unique resolving capacity of uncrosslinked polyacrylamide gels for DNA previously reported is that for heteroduplex DNA [Pulyaeva H, Zakharov SF, Garner MM, Chrambach A. Electrophoresis 15 (1994) 1095-1100] matched by crosslinked gels only in the presence of denaturants [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ. Proc Natl Acad Sci USA 90 (1993) 10 325-10 329]. A clue as to the cause of that unique resolving capacity of crosslinked polyacrylamide is provided by the finding in the present study of a single, gel concentration independent KR [retardation coefficient, d(log mobility)/d(gel concentration)] for the DNA fragments, which contrasts with the decrease of KR with gel concentration observed for crosslinked polyacrylamide across a wide concentration range [Orban L, Chrambach A. Electrophoresis 12 (1991) 241-246; Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. Since the decrease of KR with gel concentration correlates with a decrease in equivalent molecular radius [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190], it has been interpreted as being due to the transition from a random-coiled to a stretched DNA conformation upon passage through gels of increasing concentration. Since in uncrosslinked gels the decrease of KR does not occur, it is correspondingly assumed that the random-coil conformation of DNA is maintained in those gels in the investigated concentration range up to 20%. The maintenance of random-coil conformation [Tietz D, Chrambach A. Electrophoresis 14 (1993) 185-190]. The effect of denaturants in allowing for resolution of heteroduplex DNA in crosslinked gels [Peeters AV, Kotze MJ. PCR Methods Appl 4 (1994) 188-190; Ganguly A, Rock MJ, Prockop DJ, Proc Natl Acad Sci USA 90 (1993) 10 325-10 329] supports that hypothesis of the enhanced resolving power of electrophoresis in gels that maintain random-coiled DNA within the gel concentration range used.

Acrylic Resins↗