Computer-assisted evaluation of polydisperse two-dimensional gel patterns of polysaccharide-protein conjugate preparations with regard to size and net charge (original) (raw)
Related papers
Electrophoresis, 1987
ABSTRACT Two procedures of computer simulation of the electrophoretic migration of a particle through agarose gel are described which allow for: (a) characterization of gel fiber dimensions as a function of gel concentration (gel standardization), (b) determination of particle radius and the dynamics of apparent particle compressibility during passage through the standardized gel, and (c) estimation of the net charge density of a particle by calculating its mobility at 0 % gel concentration. The common model underlying these simulations is based on the extended Ogston theory which probabilistically describes the migration of a particle through a random network of inert and non-flexible fibers in terms of a “random space walk”. The first procedure, applicable to relatively rigid particles such as bacteriophages, standardizes the gel fiber on the basis of mobility values (cm/s)/(V/cm) at several gel concentrations of a single, or several, bacteriophages of known radius. Mobilities of an unknown bacteriophage are then used to simulate its physical properties. The second method, applicable to relatively non-rigid particles such as plant viruses, uses 7 polystyrene particles of known radius to standardize the gel fiber, followed by simulation of virus properties on the basis of their mobilities at several gel concentrations. The techniques described are most appropriate for deriving physical properties of particles from their nonlinear plots of log (mobility) vs. gel concentration (Ferguson plots). They have the virtue of yielding the properties of native, hydrated gel fibers and particles.
Diffusion of Macromolecules in Agarose Gels: Comparison of Linear and Globular Configurations
Biophysical Journal, 1999
The diffusion coefficients (D) of different types of macromolecules (proteins, dextrans, polymer beads, and DNA) were measured by fluorescence recovery after photobleaching (FRAP) both in solution and in 2% agarose gels to compare transport properties of these macromolecules. Diffusion measurements were conducted with concentrations low enough to avoid macromolecular interactions. For gel measurements, diffusion data were fitted according to different theories: polymer chains and spherical macromolecules were analyzed separately. As chain length increases, diffusion coefficients of DNA show a clear shift from a Rouse-like behavior (D G Х N 0 Ϫ0.5 ) to a reptational behavior (D G Х N 0 Ϫ2.0 ). The pore size, a, of a 2% agarose gel cast in a 0.1 M PBS solution was estimated. Diffusion coefficients of the proteins and the polymer beads were analyzed with the Ogston model and the effective medium model permitting the estimation of an agarose gel fiber radius and hydraulic permeability of the gels. Not only did flexible macromolecules exhibit greater mobility in the gel than did comparable-size rigid spherical particles, they also proved to be a more useful probe of available space between fibers.
Relation between the gel structure and the mobility of tracers in globular protein gels
Journal of Colloid and Interface Science, 2012
Diffusion of fluorescent-labeled dextran with different molecular weights was investigated in b-lactoglobulin (b-lg) solutions and gels over a wide range of salt and protein concentrations at pH 7 by combining confocal laser scanning microscope (CLSM) with fluorescence recovery after photobleaching (FRAP). Effects of the protein concentration, the salt concentration and the tracer size were investigated in detail. Diffusion in turbid heterogeneous gels formed at 0.2 M NaCl depended weakly on the probe size and the protein concentration and remained close to that in unheated solutions. A strong decrease of the diffusion coefficient with increasing tracer size and protein concentration was observed in more homogeneous gels formed at lower salt concentrations. Larger dextran chains were trapped in transparent gels formed at NaCl concentration below 0.1 M. The present investigation complements an earlier study of tracer diffusion of larger spherical probes in b-lg gels using multi-particle tracking.
Analysis of convex Ferguson plots in agarose gel electrophoresis by empirical computer modeling
Electrophoresis, 1986
Agarose gel electrophoresis has been shown to give rise to non-linear plots of log (mobility) vs. gel concentration of spherical viruses (Serwer, [ 11) and cellular vesicles (Gottlieb ef al. . This finding also applies to proteins as shown in this study. Considering that in the non-linear plot, the slope becomes a function of gel concentration, it is possible to determine particle properties and gel parameters by a modification of the conventional method derived from the Ogston theory for long-fiber gels. This treatment shows: a) In application to data obtained from gel electrophoresis (0.4 to 1.6 % agarose) of viruses (1 3 to 42 nm radius) and with increasing gel concentration: (i) an increase of apparent total fiber length per g agarose matrix, (ii) a reduction of apparent fiber radius and (iii) a constant fiber volume (per g matrix material) of the agarose fiber. The values of the fiber radii identify the fibers as agarose supercoils (or aggregates of it) with a radius of 20-55 nm. b) In application to data obtained from gel electrophoresis (1.2 to 8 % agarose) of proteins (1.7 to 5.8 nm radius): a fiber volume indicative of additional sieving by the agarose double helix (of known radius of 0.5-2 nm). This is in agreement with aprevious suggestion by Serwer [ 11 that proteins are able to penetrate into the double-helical network of which the agarose supercoil consists. c) The likelihood of a continuous transition from case a) to case b).
Electrophoresis, 1988
In contrast to Ferguson plots based on relative mobilities, Ferguson plots of proteins in polyacrylamide gel electrophoresis based on their absolute mobilities were found to be linear under unusual polymerization conditions which yield relatively wide gel fibers and a low total fiber length per unit weight, but not under previously and commonly used conditions. These linear Ferguson plots in gels of 1, 3 and 5% crosslinking intersect at a single gel concentration between 1 and 2 %T (M-point). It is postulated that the measure of free mobility of the proteins is the M-point, and not the intercept of their Ferguson plots with the mobility axis as assumed previously. This postulate abolishes the well-known paradoxical interpretation of the increase with %C of the linearly extrapolated intercept of the Ferguson plot with the log(mobility) axis (designated Yo) in terms of free mobility. The postulate is also compatible with the interpretation of the points of intersection of the Ferguson plots of oligomeric series of proteins at finite gel concentrations (designated μ-points) as their common free mobilities.
Macroporous gels: facts and misfacts
Journal of Chromatography A, 1995
Some extraordinary events have occurred in the last two years which might revolutionize the field of polyacrylamide gel electrophoresis. While it was widely recognized that such matrices could normally be cast with a small pore size distribution, typically of the order of a few nanometres diameter (for protein sieving), recent developments suggest that "macroporous" gels could also be produced in the domain of polyacrylamides. If constraints on chain motion are imposed during gel polymerization, large-pore structures can be grown. Such constraints can originate either from low temperatures or from the presence of preformed polymers in the gelling solution; in both instances, the growing chains are forced to "laterally aggregate" via inter-chain hydrogen bond formation. On consumption of pendant double bonds, such bundles are frozen in the three-dimensional space by permanent cross-links. As an additional development, a novel photopolymerization system is described, consisting of a cationic dye (methylene blue) and a redox couple (sodium toluene sulphinate, a reductant, and diphenyliodonium chloride, a mild oxidant). Methylene blue catalysis is characterized by a unique efficiency, ensuring >96% conversion of monomers even in aqueous-organic solvents and in presence of surfactants, which normally quench or completely inhibit the peroxodisulphate-driven reaction. In addition, methylene blue-sustained photopolymerization can be operated in the entire pH range 3-10, where most other systems fail. Perhaps the most striking novelty in the field is the appearance of a novel monomer (N-acryloylaminoethoxyethanol, AAEE) coupling a high hydrophilicity with a unique resistance to alkaline hydrolysis. Given the fact that a poly(AAEE) matrix is 500 times more stable than a poly(acrylamide) gel, while being twice as hydrophilic, it is expected that this novel chemistry will have no difficulties in replacing the old electrophoretic anticonvective media. The review ends with a glimpse at novel sieving media in capillary zone electrophoresis: polymer networks. Such media, by providing an almost infinite range of pore sizes, owing to the absence of a rigid support, allow sieving mechanisms to be operative over a wide interval of particle sizes, even up to genomic DNA. Viscous solutions of polymer networks, made with the novel poly(AAEE) chemistry, allow the repeated use of the same separation column, well above 50 injections. Silica-bound poly(AAEE) chains provide effective quenching of electrosmosis and >200 analyses by isoelectric focusing.
2006
Protein-conjugated meningitis vaccines have been developed by John Robbins, Rachel Schneerson and co-workers [1,2] for the immunization of small children, the main target group of bacterial meningitis. The physical characterization of these immunogens has been difficult, since their surface net charge is high and since the particles have a relatively large size which is in the range of intact viruses. Moreover, the size distribution of vaccine particles varies continuously over a wide range (polydisperse distribution) due to the randomizing steps in their method of preparation. When such samples are subjected to one-dimensional agarose electrophoresis, they exhibit an uninterpretable smear rather than a pattern of distinct zones (Fig. 1). However, when a second dimension is added to the separation (apparatus shown in Fig. 2), the results become interpretable: Distribution patterns are obtained which are characteristic for each vaccine. The patterns are digitized by scanning and then...
Physical and chemical properties of gels
Journal of Crystal Growth, 1999
In this work, we present a new approach using analytical and optical techniques in order to determine the physical and chemical properties of silica gel, as well as the measurement of the pore size in the network of the gel by scanning electron microscopy. The gel acupuncture technique developed by GarcmH a-Ruiz et al. (Mater. Res. Bull 28 (1993) 541) GarcmH a-Ruiz and Moreno (Acta Crystallogr. D 50 (1994) 484) was used throughout the history of crystal growth. Several experiments were done in order to evaluate the nucleation control of model proteins (thaumatin I from Thaumatococcus daniellii, lysozyme from hen egg white and catalase from bovine liver) by the porous network of the gel. Finally, it is shown how the number and the size of the crystals obtained inside X-ray capillaries is controlled by the size of the porous structure of the gel.