Low Gel Concentrations Research Articles

Gel-Shift Assays: Migrative Dissociation of a RecA−Oligonucleotide Complex during Electrophoresis in Hydroxyethylated Agarose Gels

We investigate the dynamics of a protein-DNA complex under gel-shift assay conditions, in particular how the degree of gel confinement influences the complex stability during the electrophoretic analysis. The fiber-like complex between RecA proteins and a single-stranded oligonucleotide has been studied in hydroxyethylated agarose gels with the average pore radius being between 1 and 5 times larger than the radius of the rodshaped complex. The confining effect of the gel matrix slows down the dissociation of the complex, but in addition, migrative interactions (collisions) with the gel network during the electrophoresis perturbs the complex in two ways. At low gel concentrations, the protein-DNA complex is dismantled into free RecA and oligonucleotide with a half-time of the complex that decreases with increasing field strength in accordance with a migrative mechanism. At a given field strength, the half-time increases with increasing gel concentration despite more frequent interactions, probably because of a counteracting stabilizing cage effect from the gel. At high gel concentrations, a second type of perturbation is reflected in an increasing complex velocity over time, probably caused by trains of end-to-end attached fibers being broken up by interactions with the tight gels.

Dynamics of Cellulose Whiskers in Agarose Gels. 1. Polarized Dynamic Light Scattering

The dynamics of cotton cellulose microcrystals (“whiskers”) trapped in agarose hydrogels of varying concentrations was studied by polarized dynamic light scattering. The rigid agarose network strongly heterodynes the signal of the mobile whiskers, thereby enabling the measurement of their diffusion coefficient D and their fluctuating scattered intensity Rθ. For low agarose gel concentrations, i.e., when the mesh size of the agarose network is larger than the length of the whiskers, the dynamics does not depend on the whisker content. The product of D and Rθ, which is inversely proportional to the friction coefficient, is independent of the gel concentration, indicating that the network does not hinder the whiskers. When the mesh size of the network is smaller than the length of the whiskers, the whisker content influences the dynamics and the product RθD becomes dependent on the gel concentration; i.e., frictional effects arise. Also in this condition domains with different whisker contents may develop in the gel.

Porous chitin matrices for tissue engineering: Fabrication and in vitro cytotoxic assessment

A series of porous chitin matrices were fabricated by freezing and lyophilization of chitin gels cast from a 5% N,N-dimethylacetamide (DMAc)/lithium chloride (LiCl) solvent system. The porous chitin matrices were found to have uniform pore structure in the micron range. Scanning electron microscopy (SEM) revealed that the pore size of the porous chitin matrices varied according to the freezing method prior to lyophilization. By subjecting the chitin gels to dry-ice/acetone (−38 °C), the final porous chitin matrix gave pore dimensions measuring 200–500 μm with 69% porosity. A smaller pore dimension of 100–200 μm with 61% porosity was produced when the chitin gels were frozen by liquid nitrogen (−196 °C) and 10 μm pores with 54% porosity were produced when the gels were placed in a freezer (−20 °C) for 20 min. In comparison, lower porosity structures of ca. 10% porosity were obtained from both air-dried and critical point dried chitin gels. Furthermore, a low gel concentration (< 0.5%, w/w) also produced porous morphology by vacuum drying without any freezing and lyophilization. The resulting pore properties influenced the water uptake profile of the materials in water. These porous chitin matrices are found to be non-cytotoxic and to hold promise as potential structural scaffolds for cell growth and proliferation in vitro.

Color transitional dyes as indicators of ionic boundaries in sodium dodecylsulfate polyacrylamide gel electrophoresis.

Several dyes were evaluated for their potential use as indices of ionic boundaries and pH discontinuities during polyacrylamide gel electrophoresis (PAGE). Dye selection was based largely upon color transition over the pH intervals present in the commonly used Tris-HCl-glycine buffer systems. In the absence of sodium dodecylsulfate (SDS) nitrazine yellow, cresol purple, cresol red, and phenol red had relative mobilities (relative to the chloride boundary; RC1) of 1.00 at 20% polyacrylamide concentration (% T) compared to bromophenol blue (BPB) for which RC1=0.77. The homologue of BPB, 3, 4, 5, 6-tetrabromophenolsulfonephthalein (TBPS), exhibited a useful color transition over the pH 6.6-8.2 range but was an equally poor indicator of the chloride boundary at 20% T in the absence of SDS (RC1=0.73). The RC1 of other dyes was increased in the presence of SDS, particularly the cationic dye neutral red which complexes with micellar but not monomeric SDS. In the presence of SDS, all dyes except neutral red exhibited RC1 of 1.00 at 15% T, whereas only neutral red and nitrazine yellow had RC1 values of 1.00 at lower gel concentrations. Partial characterization of SDS, in terms of RC1 and zone broadening, was performed in a system devoid of extraneous SDS in the gel or electrolytes.

Reevaluation of the Electrophoretic Migration Behavior of Soluble Globular Proteins in the Native and Detergent-Denatured States in Polyacrylamide Gels

Although sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis is widely used for estimating molecular masses of proteins, considerable uncertainty still exists both about the structure of SDS–protein complexes and about their mechanism of electrophoretic migration. In this study, soluble globular proteins, with masses of 14–200 kDa, were heat-denatured in the presence of SDS and their relative total molecular volume and net charge were estimated from Ferguson plots of electrophoretic mobility vs acrylamide concentration. Native globular protein served as standards for overall molecular size and effective radii. Results revealed at least two independent electrophoretic migration mechanisms for the SDS–protein complexes: (i) for proteins in the 14–65 kDa range at <15% acrylamide, linear Ferguson plots suggested that they migrated ideally and that their effective radii could be estimated in this manner: (ii) concave plots at higher gel concentrations, and for complexes derived from larger proteins, indicated that migration in these cases could be described by reptation theory. Migration of the large proteins at lower gel concentrations and small proteins at higher gel concentrations was not well described by either theory, representing intermediate behavior not described by these mechanisms. These data support models in which all but the smallest SDS–protein complexes adopt a necklace-like structure in which spherical micelles are distributed along the unfolded polypeptide chain. Possible relations to recent alternative models of gel electrophoresis are also discussed.

An Associative Polyelectrolyte End-Capped with Short Polystyrene Chains. Synthesis and Rheological Behavior

Polystyrene (PS) end-capped polyelectrolytes were prepared by modification of polystyrene−poly(tert-butyl acrylate)−polystyrene triblock copolymers synthesized via anionic polymerization. These amphiphilic polymers are associated in dilute aqueous solutions forming loose multimicellar clusters. At higher polymer concentrations an extensive intermicellar bridging occurs, leading to the formation of a transient network as revealed by the viscoelastic response of the system. The so-formed gel is characterized by low gel concentration, i.e., 0.2 wt %, yield stress, complex steady-shear viscosity profile, high plateau modules, and long relaxation times. These characteristics can be attributed to the highly hydrophobic character of the PS junctions and the stretched conformation of the polyelectrolyte bridges.

When can the Ogston-Morris-Rodbard-Chrambach model be applied to gel electrophoresis?

The Ogston-Morris-Rodbard-Chrambach theory of gel electrophoresis is consistent with predictions from the volume averaging method with respect to the equivalence of the accessible volume fraction to the ratios of gel mobility to free solution mobility and the gel diffusion coefficient to free solution diffusion coefficient for the limiting case of small molecule electrophoresis with low electrical fields, low gel concentrations, and nonconductive gel fibers. When these conditions are not valid, more extensive calculations are required to determine the mobility and diffusion coefficient ratios as functions of the geometry and electrical field within the gel. The volume averaging theory shows that it is important to account for the electrical conductivity properties of the fibers that make up a gel electrophoresis medium, and this aspect is consistent with early theories of transport phenomena in gel electrophoresis.

Sodium dodecyl sulfate-capillary gel electrophoresis of proteins using non-cross-linked polyacrylamide

Proteins with relative molecular masses of 14 000 to 205 000 were separated by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using non-cross-linked linear polyacrylamide gels on both coated and uncoated fused-silica capillaries. It was determined that viscosity of the acrylamide solution was a major factor affecting column stability with linear acrylamide gels. When the viscosity of the acrylamide solution reaches 100 cP, electro-osmotically driven displacement of the gels is insignificant. Uncoated capillaries provided better resolution, stability, and reproducibility than surface coated capillaries when the concentration of linear polyacrylamide was greater than 4%. At lower gel concentrations, non-cross-linked polyacrylamide is easily displaced from the columns. A calibration plot of log molecular mass vs. mobility with non-linear polyacrylamide was linear, which indicated that resolution was equivalent to that obtained with cross-linked acrylamide. Separations with model proteins indicated that baseline resolution between protein species that vary 10% in molecular mass can be achieved.

Solute diffusion in biocatalyst gel beads containing biocatalysis and other additives

Biocatalyst beads can be produced by the entrapment of microorganisms or enzymes into a stabilized hydrocolloidal gel such as alginate or carrageenan. In order to determine and predict the effectiveness of such catalytic material, it will be necessary to know the mass transport properties of substrates and products within the gel beads, especially where there are high concentrations of the biocatalyst or other additives. Diffusion of several solutes within the beads has been studied by measuring the transport of solutes to and from a well-stirred solution of limited volume. Tests have been performed with biocatalyst beads made from alginate and carrageenan covering a range of bead diameter, gel concentration, concentration of biocatalyst and other additives, and solute molecular weight. At low gel concentration with entrapped biocatalyst, a solute of low molecular weight has a diffusion coefficient approaching that measured in water. However, with increased gel concentration, and especially with high loadings of microorganisms and other additives, the diffusion coefficients are significantly decreased. An empirical relationship was developed for the diffusion coefficient as a function of microbial loading.

Gel electrophoretic analysis of DNA branched junctions

Gel electrophoresis has provided much of the detailed information we have about the properties of DNA junctions, stable branched molecules formed from oligonucleotide or polynucleotide strands. Here we review these applications, and present the results of an electrophoretic investigation of conformationally restricted junctions formed by covalently connecting two different pairs of strands in a junction with four arms. Native gel electrophoresis is employed to establish the formation and stoichiometry of the multistrand complexes. Ferguson analysis of native gel mobility shows that junctions have retardation coefficients that are distinct from those of linear DNA duplexes. Denaturing gel electrophoresis is the primary tool for characterizing junctions that have been covalently linked together to form both linear and macrocyclic oligomers of junctions (oligojunctions). Radioactively labelled strands enable one to monitor the progress of the ligation reaction: both linear and closed cyclic molecules result, and these can be distinguished by applying Ferguson analysis to denaturing gels. Combinations of exonuclease III, restriction enzymes and sequencing reactions have been applied to oligojunction molecules, and the results are all analyzed on denaturing gels. Junctions containing intramolecular "tethers" that restrict the conformation freedom of the complex comprise a new system for analyzing the conformations of branched molecules. In these tethered junctions, the ability of arms to move relative to each other is restricted substantially by covalently connecting pairs of arms in the original complex with short, flexible loops. The two tethers used here constrain the helical domains of the structure to be roughly parallel or anti-parallel. In this article, we use Ferguson analysis to compare two tethered junctions with an untethered junction. At high gel concentrations, the mobility of the untethered complex is found to be closer to that of the molecule tethered anti-parallel than to the one tethered parallel. Curvature in the Ferguson plots for all three of these junctions is detected over a range of compositions. At low gel concentrations, differences in electrophoretic mobility persist, suggesting that the untethered junction differs in charge as well as conformational freedom from the tethered analogs. We expect that studies of this kind will be able to define the conformational repertoire of junctions of different kinds, and to explore the effects of electrophoresis on these states.

Discontinuous buffer systems optimized for the agarose gel electrophoresis of subcellular particles

Discontinuous buffer systems operative between pH 5.7 and 7.4, 0 degrees C, were generated, which are characterized by more rapidly displaced moving boundaries than applied previously. These allow one to resolve subcellular particles relatively rapidly and at relatively low agarose gel concentrations. A commercial mixture of DNA restriction fragments pre-stained with ethidium bromide was found to be a suitable tracking dye for these boundaries.

Laser light scattering study of polyacrylamide gels

AbstractLaser light scattering experiments with polyacrylamide gels containing diffusing bovine plasma albumin are described. Complete heterodyne beating has not been assumed, and an account is given of a novel method for finding the degree of spectral broadening by scanning angles of scatter while measuring the temporal autocorrelation function. Results obtained by this method for polyacrylamide gels and un‐cross‐linked solutions in the absence of diffusing protein, are compared with those of other authors. In the presence of the protein there is a complex interaction between the diffusion coefficient of the protein and the coefficient associated with the diffusing fluctuations in segment density of polyacrylamide. The diffusing protein dominates at low gel concentrations, and the gel density fluctuations dominates at high gel concentrations. It is proposed that the protein preferentially occupies the less dense regions of the gel, so that at low gel concentrations, where it diffuses faster than the gel fluctuations, it tends to cancel them out, whereas at high gel concentrations where the protein is highly impeded by the gel structure, it tends to be carried along by the gel density fluctuations.

The Diffusion of Compact Macromolecules Within Hydrogels

Studies of the diffusion of globular proteins and dextran fractions within polysaccharide and poly-acryamide gels by laser light scattering are described. Gels of stiff polysaccharide chains scatter light which exhibits little spectral broadening, since apart from a small amount of structural relaxation they are almost stationary at a molecular level. Well defined diffusion coefficient of compact macromolecules within these gels can be measured. However, gels of relatively flexible polymers such as polyacrylamide are highly mobile at a molecular level, and scatter light which is spectrally broadened by the diffusion of fluctuations in acrylamide concentration. In this case there is a complex interaction between the two diffusion coefficients, with the diffusing compact macromolecules dominating at low gel concentrations, and the diffusing gel fluctuations dominating at high gel concentrations where they tend to control the movement of the compact macromolecules.

Use of CH 3HgOH-agarose gels for the electrophoresis of heterogeneous nuclear RNA and messenger RNA from mammalian cells

The size distribution of labeled hnRNA and mRNA from Hela cells and mouse Ehrlich ascites cells has been analyzed by gel electrophoresis in the presence of the denaturing agent CH 3HgOH. Up to 43% of the mass of total hnRNA consisted of molecules at least 28 kilobases in size, provided this RNA had been extracted under conditions minimizing degradation. Ten to sixteen percent of the mass of polyadenylated hnRNA consisted of molecules larger than 13 kilobases, although the average molecular weight was less than that of total hnRNA. In comparison, more than 90% of the mass of labeled mRNA (total or polyadenylated) consisted of molecules smaller than 5.5 kilobases. A linear relationship was observed between appropriate functions of molecular weight and mobility for RNA molecules up to at least 13 kilobases in size, provided a low gel concentration and voltage gradient were employed. Increasing these parameters resulted in anomalously fast migration of large RNA molecules. Electrophoretic profiles were not significantly altered when the amount of poly(A) hnRNA was increased from about 1 to 50 μg per cylindrical gel. Thus CH 3HgOH-agarose gel electrophoresis can be used preparatively for the fractionation of hnRNA. Aggregates containing labeled mRNA (formed in vitro in the presence of excess nuclear RNA) were dissociated by CH 3HgOH. Partial degradation of high molecular weight RNA was observed after prolonged exposure to CH 3HgOH.

Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffers using multiphasic buffer systems: Properties of the stack, valid Rf− measurement, and optimized procedure

SDS-proteins can be stacked in sharp starting zones in SDS-PAGE in multiphasic buffer systems, Stacking of SDS-proteins has been possible with a lower stacking limit of up to 0.300, above neutral pH in “nonrestrictive” gels. Under the same conditions, SDS is stacked. Stacking of SDS, derived from both the SDS content of the sample and the upper buffer, broadens the stack in proportion to SDS load and duration of electrophoresis. Such broadening produces an inordinately wide starting zone in the stacking gel, leading to impaired resolution, unless one limits the SDS load. In the resolving gel, this zone broadening makes the trailing edge of the stack, and the tracking dye migrating there [e.g., bromphenol blue at low gel concentration (% T)] unsuitable as a valid reference for R f . However, the leading edge of the stack and the tracking dye migrating there (e.g., pyronin-Y-SDS at low % T) are a valid R f reference. At high % T, both SDS and pyronin-Y-SDS unstack, whereas bromphenol blue now moves to the leading edge of the stack and becomes, for that % T range, a valid reference of R f . An optimized system for SDS-PAGE is described which contains no SDS in the gel, 0.03% SDS in the upper buffer, 150 μg or less SDS in the sample, and a single set of gel buffer constituents in stacking and resolving gels. Valid R f measurement is a requisite for linear Ferguson plots (log R f vs % T). Since the slope of these plots, K R , is a measure of molecular weight the procedure of SDS-PAGE proposed in this study should provide improved reliability of melecular weight estimates.

Unusual migration profiles of Japanese encephalitis virus structural polypeptides in SDS-polyacrylamide gel electrophoresis.

The migration profiles of JEV polypeptides in SDS-PAGE are influenced by electrophoretic conditions. The estimated molecular weights of N and M became smaller when lower gel concentration and longer electrophoretic run were adopted.

On the mechanism of the molecular-sieve effect in polyacrylamide gel electrophoresis

A model for the mechanism of the molecular-sieve effect in polyacrylamide gel electrophoresis is presented for low gel concentrations. From mobility measurements at different gel concentrations, the model gives radii for protein molecules which are in good agreement with the values calculated from the known molecular weights.

DIFFUSION OF TRITIATED WATER (H3H1O16) IN AGAR GEL AND WATER

The diffusion coefficient of H3H1O16 at 25 deg C was measured at four low agar gel concentrations and the coefficient in liquid water obtained by extrapolating to zero gel concentration. The resulting value is D = 2.41 plus or minus 0.055 x 10-5 cm2 sec-1, which agrees with the value of Wang et al.