Biomedical subjects

S P Radko

Publications and source records attributed to S P Radko.

26 records · Page 2Linked to original sources

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↗

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

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↗

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↗

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

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↗