Diffusion of Macromolecules in Agarose Gels: Comparison of Linear and Globular Configurations (original) (raw)
Related papers
Size Effects on Diffusion Processes within Agarose Gels
Biophysical Journal, 2004
To investigate diffusion processes in agarose gel, nanoparticles with sizes in the range between 1 and 140 nm have been tested by means of fluorescence correlation spectroscopy. Understanding the diffusion properties in agarose gels is interesting, because such gels are good models for microbial biofilms and cells cytoplasm. The fluorescence correlation spectroscopy technique is very useful for such investigations due to its high sensitivity and selectivity, its excellent spatial resolution compared to the pore size of the gel, and its ability to probe a wide range of sizes of diffusing nanoparticles. The largest hydrodynamic radius (R c) of trapped particles that displayed local mobility was estimated to be 70 nm for a 1.5% agarose gel. The results showed that diffusion of particles in agarose gel is anomalous, with a diverging fractal dimension of diffusion when the large particles become entrapped in the pores of the gel. The latter situation occurs when the reduced size (R A /R c) of the diffusing particle, A, is .0.4. Variations of the fractal exponent of diffusion (d w) with the reduced particle size were in agreement with three-dimensional Monte Carlo simulations in porous media. Nonetheless, a systematic offset of d w was observed in real systems and was attributed to weak nonelastic interactions between the diffusing particles and polymer fibers, which was not considered in the Monte Carlo simulations.
Macromolecules, 2000
Laser scanning confocal microscopy combined with fluorescence recovery after photobleaching is an effective tool to measure the diffusion coefficients of macromolecules in cross-linked hydrogels and polymer solutions. In this study, the effects of enzyme treatment on the diffusion of macromolecules (FITC-dextran) in guar solutions and titanium-guar hydrogels are examined. Enzyme treatment with -mannanase, a polymer backbone cleaving enzyme, quickly increases the diffusion coefficient of the probe molecules in both solutions and hydrogels to that in water. Enzyme treatment of guar solutions and hydrogels with R-galactosidase, a side chain cleaving enzyme, displays a unique behavior due to changes in the fine structure of guar. The removal of galactose branches from the mannan backbone of guar creates additional hyperentanglements (i.e., cross-links), which reduce the water holding capacity of guar and induce syneresis. If the depth at which the diffusion coefficient is measured remains constant, a minimum is observed in the diffusion coefficient as R-galactosidase enzyme treatment time increases. At the site of measurement, the sample changes from a homogeneous guar system to a phase-separated polymer-rich hydrogel and finally to a dilute polymer phase as the polymer-rich hydrogel phase precipitates below the site of measurement. The diffusion coefficient in the dilute polymer phase increases to that in water, while the diffusion coefficient in the hydrogel phase continues to decrease to a value of approximately 6 × 10 -8 cm 2 /s. 10.
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.
ELECTROPHORESIS, 2006
Porous hydrogels such as agarose are commonly used to analyze DNA and watersoluble proteins by electrophoresis. More recently lyotropic liquid crystals, such as the diamond cubic phase formed by the lipid monoolein and water, has become a new type of well-defined porous structure of interest for both hydrophilic and amphiphilic analytes. Here we compare these two types of matrixes by investigating the nature of retardation they confer to an oligonucleotide that migrates in their respective aqueous phases. The retardation for a 25-mer oligonucleotide was found to be about 35-fold stronger in the cubic phase than in an agarose hydrogel modified to have the same average pore size. According to modelling, the strong retardation is primarily due to the fact that hydrodynamic interaction with the continuous monoolein membrane is a stronger source of friction than the steric interactions (collisions) with discrete gel fibres. A secondary effect is that the regular liquid crystal has a narrower pore-size distribution than the random network of the agarose gel. In agreement with experiments, these two effects together predict that the retardation in the cubic phase is a 30-fold stronger than in an agarose gel with the same average pore radius.
ELECTROPHORESIS, 2002
Electrophoretic mobilities of DNA molecules ranging in length from 200 to 48 502 base pairs (bp) were measured in agarose gels with concentrations T = 0.5% to 1.3% at electric fields from E = 0.71 to 5.0 V/cm. This broad data set determines a range of conditions over which the new interpolation equation m(L) = (b1a(11exp(-L/g)) -1 can be used to relate mobility to length with high accuracy. Mobility data were fit with w 2 . 0.999 for all gel concentrations and fields ranging from 2.5 to 5 V/cm, and for lower fields at low gel concentrations. Analyses using so-called reptation plots (Rousseau, J., Drouin, G., Slater, G. W., Phys. Rev. Lett. 1997, 79, 1945-1948 indicate that this simple exponential relation is obeyed well when there is a smooth transition from the Ogston sieving regime to the reptation regime with increasing DNA length. Deviations from this equation occur when DNA migration is hindered, apparently by entropic-trapping, which is favored at low fields and high gel concentrations in the ranges examined.
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.
Polymer, 1982
The diffusion of solutes in gels is comprehensively reviewed. Because it has been a source of confusion, precise definitions of the gel diffusion coefficient are presented and discussed. Theories as to the effect of the gel substance on the course of diffusion are critically evaluated. These include the obstruction effect, hydrodynamic drag and other frictional couplings, alteration of solvent properties and (for homogeneous gels) the free volume theory. A large proportion of the data on diffusion in gels to be found in the literature is displayed, with the exception of those systems where binding of the solute is a major factor. The success of the theories in accounting for these results is examined. It is concluded that for heterogeneous gels the obstruction effect is prevalent, for organic solvent-polymer systems the free volume theory has had some success while diffusion of both macromolecules and micromolecular solutes in homogeneous gels is not well understood and deserves more experimental effort.
Mobility models for stiff and flexible macromolecules in dilute gels
Biopolymers, 1991
The effective sphere approximation for modeling electrophoretic transport of macromolecules in highly porous gels (the “Ogston model”) is examined, and contrasted with similar mobility models for stiff and flexible solutes. Calculation of segmental depletion near gel obstacles of various shapes demonstrates the limited applicability of the effective sphere approach. For highly flexible chains, both theory and experiment reveal a nonunique mapping between mobility and molecular size when the molecular radius is comparable to that of gel fibers. Turning to mobility behavior in more concentrated gels, neither flexible or stiff macromolecules behave as spheres; for the particular case of flexible chains, the presence of entropic barriers in concentrated gels can be understood in terms of a simple random planes model for the gel structure.
Motion of large DNA molecules traveling from solution to gel under steady field
Journal of Polymer Science Part B: Polymer Physics, 1996
Direct observation of individual T4 DNA molecules at the interface between gel and pure solvent under steady electric field was carried out with fluorescence microscopy. Statistical analyses of the image data show that (1) when a DNA arrives at the gel surface, it remains there for a short time before the DNA stretches its arms to enter the gel, and (2) the time spent on the gel surface increases with an increase of the agarose persentage in the gel.