Increasing Gel Concentration Research Articles

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.

Characterizing the gloss properties of hydrocolloid films

Hydrocolloid films were prepared by two methods, namely dehydration of thin layers of gels and of gum solutions. Gelatin gel films containing greater amounts of dry matter than all other prepared films provided by far the highest gloss values. Increase in gelatin concentration in the gels resulted in an increase in the mean gloss of the dry films in a linear fashion. Roughness measurements and scanning electron microscope (SEM) micrographs indicated that the gelatin films possessed very smooth surfaces. Similar to gelatin, increase in concentration of gellan resulted in an increase in gloss of resultant films. In direct contrast, mean gloss of both agar and agarose gel films decreased with increasing gel concentration. These trends may be due to the increase in roughness of films with increasing gel concentration, which in the case of agar was also noticeable in the SEM micrographs. Xanthan films provided the most glossy appearance of those prepared by drying of gum solutions, followed closely by alginate. Both exhibited the same trend of mean gloss increasing with gum concentration, similar to gelatin. In both instances, this behaviour could not be related to roughness, which also increased with gum concentration.

Sequestration StabilizeslacRepressor–DNA Complexes during Gel Electrophoresis

The gel electrophoresis mobility shift assay is widely used for both qualitative and quantitative characterization of protein–nucleic acid interactions. Often it is found that protein–nucleic acid complexes persist within gels for much longer than would be expected on the basis of their free solution lifetimes. Excluded volume and matrix-interaction mechanisms have been proposed to account for the enhanced stabilities of complexes within gels. To test these mechanisms, we have investigated the influences of gel composition and concentration on the pseudo first-order dissociation kinetics of complexes containing theEscherichia colilactose (lac) repressor protein and lactose promoter DNA. In both polyacrylamide and agarose gels, dissociation rates were slower than those in free solution and decreased with increasing gel concentration. This result is inconsistent with mechanisms of stabilization that require specific interactions with the gel matrix. Under standard reaction conditions, free solution values ofkdisswere proportional to [DNA]0.83±0.11, while in 10% polyacrylamide gelskdissvalues were proportional to [DNA]0.48±0.09. These results suggest that the lifetimes oflacrepressor–DNA complexes in free solution are limited by their encounter frequency with molecules of DNA or with protein–DNA complexes; some or all of the stabilization observed in gels may be due to a reduction in this frequency.

DNA mobility anomalies are determined primarily by polyacrylamide gel concentration, not gel pore size.

The dependence of DNA mobility anomalies on gel pore size has been studied in polyacrylamide gels with a wide variety of compositions, using molecular weight ladders containing multiple copies of normal (12B) and anomalously slowly migrating (12A) 147-base pair restriction fragments from plasmid pBR322 as the migrating probe molecules. If the gel pore size is increased by decreasing the total acrylamide concentration (%T) at constant cross-linker ratio (%C), the usual method of increasing gel pore size, the mobility anomalies decrease with increasing gel pore radius as though the 12A multimers were retarded by a sieving mechanism. However, the decrease in the mobility anomalies is independent of whether the apparent gel pore radius is larger or smaller than the DNA radius of gyration, suggesting that gel pore size is not the controlling variable. If the acrylamide concentration is held constant and the gel pore size is increased by decreasing %C at constant %T, the mobility anomalies of the largest 12A multimers (6 mers and higher) decrease with increasing gel pore radius, because of sieving effects, until the effective gel pore radius becomes approximately equal to the DNA radius of gyration, after which the mobility anomalies level off and become independent of gel pore size. The mobility anomalies exhibited by 5-mers and smaller multimers of fragment 12A are independent of gel pore radius in all gels with constant %T. Similar results are observed with a molecular weight ladder containing phased A-tracts from the kinetoplast bending locus. Since the anomalous electrophoretic mobilities depend primarily on the total acrylamide concentration in the gel, and not on the apparent gel pore radius, increases in the magnitude of the mobility anomalies with increasing gel concentration (and decreasing gel pore radius) cannot be taken as evidence for DNA curvature.

Diffusion of proteins and nonionic micelles in agarose gels by holographic interferometry

AbstractDiffusion coefficients measured by holographic interferometry are presented for two globular proteins and three nonionic surfactant species in agarose gels. The cloud points for the surfactants in agarose solutions are also given. In all cases, rates of diffusion in the gel are smaller than those in bulk solution, with the hindering effect of the gel increasing both with increasing gel concentration and with increasing solute size. It is shown that the diffusion rate of the surfactant micelles is very similar to that of proteins having similar hydrodynamic radii; it would therefore appear that the size and shape of the micelles in the solution and gel phases are similar. In addition, the measured rates of hindered diffusion agree very well with rates predicted by a rigorous hydrodynamic theory in which the solutes are modeled as hard spheres and the gel fibers are modeled as straight, cylindrical fibers. Hence, for the range of conditions considered, rates of hindered diffusion for both the globular proteins and the micellar species are determined primarily by hard‐sphere hydrodynamic and steric interactions between the solute and the gel matrix.

Period Times and Helix Alignment during the Cyclic Migration of DNA in Electrophoresis Gels Studied with Fluorescence Microscopy

Fluorescence microscopy of YOYO-stained DNA molecules has been used to study the periodicity of the cyclic migration during constant field electrophoresis in agarose gels. The distribution of period times is asymmetric and wide, with a ratio of standard deviation to mean value of 0.50 for fields between 3 and 24 V/cm. The average period time decreases with the electric field strength, E, as E -1.2 , and increases with increasing molecular weight (38-740 kbp) and increasing gel concentration (0.5-2%). The period times for T2 DNA (164 kbp) are in good agreement with the time to the orientation undershoot, as measured in a separate linear dichroism study of YOYO-stained DNA undergoing ordinary zone electrophoresis (Carlsson et al., manuscript in preparation). This shows that linear dichroism can be used to quantify the period time of the cyclic motion and to investigate effects of changes in DNA or gel properties. The cyclic conformational changes in the global DNA conformation during migration have been characterized in terms of the apparent length L μ and end-to-end distance h μ of the molecular paths in the gel. The formation of U conformations dominates the cycle time, since it is slow compared to U slipping and retraction. The maximum and cycle-averaged orientation factors for the apparent molecular path are evaluated from the images to be 0.70 and 0.67 ± 0.03, respectively, for T2 DNA at 8 V/cm in 1% agarose. From these values the degree of helix orientation below the level of microscope resolution could be estimated by comparison with the total orientation factor for the DNA helix as measured with linear dichroism. The maximum and cycle-averaged orientation factor for the helix with respect to the global path were 0.27 and 0.22 ± 0.01, respectively. These orientation factor values demonstrate (1) a very high degree of field alignment of the global path and (2) a degree of stretching of the helix along that path which is considerable, and stronger than expected for a wormlike chain spanning a cavity with the average pore size. If the imaged path corresponds to the reptation tube, the estimated friction coefficient for motion along the tube is found to be close to the value in the buffer.

Physicochemical studies using amylose as an in vitro model for resistant starch

The effects of hydrolysing amylose gels and films with porcine pancreatic alpha-amylase were studied in order to construct an in vitro model of resistant starch. Both gels and films proved to be partially resistant to hydrolysis, their susceptibility decreasing with increasing substrate concentration. Storage of gels for 1 day resulted in levels of crystallinity from 6 to 12%. Hydrolysis of these gels produced significant increases (from 272 to 557%) in crystallinity in all cases. The amount of resistant material remaining after hydrolysis increased (from 33% to 50%) with increasing gel concentration. Storage of amylose gels for 7 days resulted in levels of crystallinity of 28 to 35%. Hydrolysis of these gels led to increases in crystallinity, but to a lesser extent (from 148 to 175%) than before. The amount of resistant material remaining after hydrolysis increased (from 41 to 50%) with increasing gel concentration. Size-exclusion chromatography of resistant material from hydrolysed films indicated the presence of a high molecular weight component corresponding to undigested amylose, in addition to a low molecular weight component corresponding to partially digested amylose. Chromatograms of resistant material from both gels stored for 1 day and those stored for 7 days were almost identical and contained only a low molecular weight component with no sign of undigested amylose. X-ray line-broadening analysis showed the average sizes of the crystallites in the resistant material produced by hydrolysis of gels stored for both 1 day and 7 days to be approximately equal. The ability of the enzyme to diffuse into its substrate played a major part in the resistance to hydrolysis of amylose films, but had a much less important role in the resistance of gels. Not only crystalline, but also amorphous portions of amylose gels, were found to be resistant to enzymic hydrolysis. The resistant portion of amylose gels was modelled in vitro with a view to explaining the structure of resistant starch in vivo.

Capillary gel electrophoresis separation of high-mannose type oligosaccharides derivatized by 1-aminopyrene-3,6,8-trisulfonic acid.

In this paper we report the capillary gel electrophoresis separation of 1-aminopyrene-3,6,8-trisulfonic acid (APTS) labeled oligosaccharides, released enzymatically from bovine pancreatic ribonuclease B. The released and labeled high-mannose structures were identified by spiking the separated peaks with the appropriate commercially available individual oligosaccharides. Baseline separation of the three positional isomers of the mannose-7 and mannose-8 oligosaccharides was attained. Comparison of the electrophoretic mobilities of the high-mannose type branched carbohydrates to the linear molecules of maltooligosaccharides (glucose oligomers) have been shown using different gel concentrations in the running buffer system. We observed that increasing gel concentration in the running buffer causes an increase in the relative mobility values of the high-mannose type carbohydrate molecules compared to the linear glucose oligomers. Analysis of our data indicated that this increase in relative migration time was not due to sieving, but seemed to be related to the mannose content and hydrodynamic volume of the branched glycans as well as to the viscosity of the separation medium.

Band width measurement in automated gel electrophoresis apparatus: DNA dispersion in a discontinuous system and in a single buffer.

Recent commercial introduction of automated gel electrophoresis apparatus allows for band width measurements during electrophoresis and therefore promises to open up the exploitation of band width and shape for the physical characterization of charged macromolecules in the same manner in which to date quantitative gel electrophoresis had exploited electrophoretic mobility at multiple gel concentrations. The measurements demonstrate decreased band width and therefore increased resolving power for a discontinuous buffer system compared to Tris-borate EDTA buffer. The dispersion coefficients (D' = (sigma 2-sigma 2o)/t) of homogeneous DNA components appear to decrease with gel concentration when either the field strength or the DNA length is small, and increase with gel concentration when these are large. This contrasting response of D' to increasing gel concentration is presumably due to DNA stretching, which increases in proportion to DNA length and field strength, and to the progressive orientation of agarose with increasing field strength.

Effects on fluid and Na+ flux of varying luminal hydraulic resistance in rat colon in vivo.

1. A new method of measuring fluid and ionic movements and the dehydrating power of the colon in vivo is described. A range of agarose gel cylinders, with calibrated hydraulic conductivities (Lp), were inserted into the lumen of the descending colon of anaesthetized rats. Fluxes of fluid, Na+ and K+ out of the gels were measured over a period of 60-110 min. 2. Fluid absorption by the colon from 2.5% agarose gels was not slower than from solution without gel. Fluid absorption was inhibited by 66% when the agarose concentration was raised to 10%. In contrast 2.5% agarose gels caused a 73% (P < 0.001) reduction in water flow from rat ileum. 3. Increasing gel concentration to 10% or above caused the absorbate from the gels to become hypertonic (P < 0.001). 4. The measured suction pressure applied by the colonic hypertonic absorbate to the gels increased from 44 +/- 2.3 cmH2O (n = 23) with 2.5% agarose gels to 6713 +/- 960 cmH2O (n = 13) with 15% (P < 0.001). 5. Deoxycholate (2 mM) produced a decrease in fluid and Na+ absorption and reduced the suction pressure and power exerted by the colon.

Characterization of the electrophoretic properties of nucleosome core particles by transverse polyacrylamide pore gradient gel electrophoresis.

Transverse pore gradient gel electrophoresis, previously applied to bent DNA, has extended the usefulness of the gel retardation assay in two ways: (i) by differentiating between different DNA conformations; (ii) by providing information regarding the physical properties of DNA. In the present study, similarly extended information is obtained with regard to a well-characterized DNA-protein complex, the chicken erythrocyte nucleosome core particle. (i) The winding of DNA around the protein core constrains the DNA which renders its Ferguson curve (migration distance vs. gel concentration) similar to that of kinetoplast DNA, i.e. it intersects sharply with the Ferguson curves of linear DNA standards. By contrast, the deproteinized nucleosome DNA exhibits a Ferguson curve similar to linear standards of the same length. (ii) Interpretation of the Ferguson curve based on a mathematical model shows that the nucleosome exhibits a linear Ferguson plot [log(mobility) vs. gel concentration]. This is similar to and characteristic of spherical proteins, contrasting with the concave plot typical for linear and bent DNA. (iii) The effective size of the nucleosome, evaluated in terms of an "equivalent sphere" (i.e. a hypothetical spherical particle with a radius, Res, having the same electrophoretic mobility as DNA for a particular set of experimental conditions), remains invariant across the gel concentration range of 3-9%T. This is similar to proteins and bacteriophages and contrasts with the progressive decline of Res with increasing gel concentration observed for linear DNA and the deproteinized nucleosomal DNA.

Effect of temperature, gel concentration and cytokinins on vitrification of Olearia microdisca (J.M. Black) in vitro shoot cultures

The interaction between gel concentration, cytokinin levels and temperature in vitrification of Olearia microdisca in vitro has been investigated. Vitrified plants displayed tissue hypertrophy and reduced lignification. Cytokinin in the medium was essential for vitrification. Increasing gel concentration or reducing temperature reduced the vitrification in the presence of 20 µM BA. The observed responses were consistent with indirect effects on BA uptake. Alternatively an indirect effect of BA on shoot multiplication rate and hence on lignification of cell walls may be involved.

Determination of the effective diffusion coefficient of oxygen in gel materials in relation to gel concentration

The effective diffusion coefficient of oxygen, IDe, was determined in different gel support materials (calcium alginate, κ-carrageenan, gellan gum, agar and agarose) which are generally used for immobilization of cells. The method used was based upon fitting Crank's model on the experimental data. The model describes the solute diffusion from a well-stirred solution into gel beads which are initially free of solute. The effect of the gel concentration on IDe of oxygen in the gel was investigated. The results showed a decreasing IDe for both agar and agarose at increasing gel concentration. In case of calcium alginate and gellan gum, a maximum in IDe at the intermediate gel concentration was observed. It is hypothesized that this phenomenon is due to a changing gelpore structure at increasing gel concentrations. The IDe of oxygen in calcium alginate, κ-carrageenan and gellan gum varied from 1.5*10−9 to 2.1*10−9 m2s−1 in the gel concentration range of 0.5 to 5% (w/v).

Analysis of convex Ferguson plots in agarose gel electrophoresis by empirical computer modeling

AbstractAgarose gel electrophoresis has been shown to give rise to non‐linear plots of log (mobility) vs. gel concentration of spherical viruses (Serwer,[1]) and cellular vesicles (Gottlieb et al.[2]). 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 (13 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 a previous suggestion by Serwer [1] 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).

Self-diffusion of Co 2+ ions in CoCl 2 and CoSO 4, and electrolyte-diffusion of CoSO 4 and ZnSO 4 in agar gel medium

Self-diffusion of Co 2+ ions in CoCl 2 and CoSO 4 in the concentration range of 10 −5 to 0.25 M is studied in 1% agar gel at 25°C using zone-diffusion technique. The divergence observed between theoretical and experimental values of diffusion coefficients in both systems is explained on the basis of various types of interactions occurring in the system. The applicability of the transition state theory to the diffusion of ZnSO 4 in agar gel medium is tested by varying the temperature of the system as well as the gel concentration. The activation energy and D 0 value decrease with increasing gel concentration, in agreement with the theory. The obstruction effect determined in terms of α-value, describing the relative lowering of the diffusion coefficient in gel medium in comparison with the pure solution, in the diffusion of ZnSO 4 is found to be independent of temperature. The effect of electrolyte concentration on obstruction effect and on activation energy ( E) for diffusion of CoSO 4 is also examined. The decrease in α and E values with electrolyte concentration is explained by considering the competitive hydration between ions and agar molecules and Wang's model respectively.

Effect of the electric field on the apparent mobility of large DNA fragments in agarose gels.

AbstractThe electrophoresis of a series of DNA fragments ranging in size from 0.5 to 12 kilobase pairs, has been studied as a function of agarose gel concentration and electric field strength. The apparent mobility of all fragments decreased with decreasing electric field strength and with increasing gel concentration. When extrapolated to zero electric field strength and zero agarose concentration, the apparent mobility of all DNA fragments extrapolated to a common value (2.0 ± 0.1) × 10−4 cm2/V s. The square roots of the retardation coefficients of the various fragments were found to be linearly related to the root‐mean‐square radii of gyration of the fragments, as predicted by pore‐size distribution theory. As predicted by reptation theory, the molecular weights of the various fragments were found to be linearly related to the reciprocal of the apparent mobilities. An equation is given for estimating the apparent pore size of agarose gels between 0.25 and 1.5% in concentration.

Self-diffusion of chromate ion in agar gel

The self-diffusion coefficient of the chromate ion is determined in agar gel media over the concentration range 10 −5 to 0.25 M at 25°C. Comparison of the experimental results and the theoretical values indicates failure in the application of Onsager-Fuoss theory to the ion diffusion in swollen gel system at all the concentrations studied. The observed results are explained on the basis of water-gel, ion-water and ion-ion interactions and the obstruction effect. Diffusion rates are also determined at various gel percentages and at different temperatures, keeping the electrolyte concentration constant. The obstruction effect is found to depend on the concentration of the electrolyte and the activation energy is found to decrease with increasing gel concentration. The results are discussed in the light of transition state theory of diffusion.

Multiple forms of glucocorticosteroid receptors in the neural retina of chick embryo, revealed by polyacrylamide gel electrophoresis

The glucocortiocoid receptors in the cytosol of neural retina of the 15-day chick embryo were analyzed by quantitative polyacrylamide gel electrophoresis. Maintenance of the triamcinolone acetonide (TA)-receptor complexes under conditions of electrophoretic analysis is dependent on temperatures not exceeding −2 °C and is favored by low ionic strength, but is relatively insensitive to changes in pH between 5 and 10. Polyacrylamide gel electrophoresis in highly crosslinked Resolving Gels (15% crosslinking with N,N′-diallyltartardiamide) at low wattage and under temperature control at −2 °C, allowed for detection and partial characterization of over 80% of the specific TA-binding activity of the tissue. One form of the glucocorticoid receptor, designated as complex II, was found to have a molecular weight ( M r) of 175,000. In addition, specifically bound TA was found in a multimillion M r aggregate which was unable to enter gels of any concentration investigated and has been designated TA-complex I. The ratio of complex I/complex II increased with increasing gel concentration, indicating physical or chemical interaction between II and I. A polyacrylamide gel electrophoresis rerun of isolated TA-complex II gave rise to two smaller TA-binding species: Component B, of M r 108,000 and component A, a relatively fast migrating molecule which could not be characterized under the conditions used. The ratio of B A appeared constant and close to 2, suggesting that A and B may be significant structural elements of complex II. Polyacrylamide gel electrophoresis of isolated TA-complex I gave rise to component C of M r 60,000, but not to components A or B. Components A and B associated to a large M r complex, designated as I′, which was revealed to an extent directly proportional to gel concentration. Similarly, component C aggregated to I″, as evidenced at elevated gel concentrations. In conclusion, it has been possible to define by gel electrophoresis three of the molecular species (A, B, and C) that comprise the glucocorticoid receptor, and the possible relationships between them.

A sedimentation study of gel systems

When an agar gel was subjected to a high centrifugal field, a well-defined gel/solvent in terface formed, and moved in the direction of the field, sometimes with a self-sharpening effect. Little non-gelling solute occurred. At high centrifugal fields, there was no observable time lag between application of the field and motion of the boundary. A plot of log x against t ( x being distance of the interface from the centre of rotation and t the time) was almost linear initially but the slope decreased progressively and the curve approached a well-defined limiting value log x ∞ appropriate to the field and initial concentration. This behaviour is probably due to the continuously increasing gel concentration which is caused as the gelling solute is confined to a progressively smaller volume between the gel interface and the bottom of the cell. When no further motion occurs, equilibrium has been attained, a tall levels of the cell, between the partial hydrostatic pressure involved and the swelling pressure of the gels. At low fields, a considerable time lag (induction period), observed between application of the field and motion of the boundary, probably indicates the need for a considerable break ­ down of structure before flow can occur. A plot of initial slope (d log x /d t ) against the square of the rotational speed is linear within experimental error and passes through the origin. The yield point for gels may be associated with the occurrence of the induction period. On the basis of a crude lattice model, the mean pore size for a 1 % agar gel was calculated to be 70 to 100 Å, in fair agreement with previous estimates. Gelatin gels behave similarly to agar in the general features of the curves of log x against t but a much higher proportion of gelatin is observable in true solution (30 to 40% ), sedimenting more slowly than the gel interface.

Electrophoresis in synthetic gels. II. Effect of gel concentration

Increasing the concentration of acrylamide gel decreases the absolute electrophoretic mobility of hemoglobins, egg albumins, and serum albumin polymers. In a series of hemoglobins, the relative mobility of any two is unaffected by changes in gel concentration; the same is true of the principal components of egg albumin. However, the mobilities of albumin polymers (relative to the monomer) decreases linearly with increasing gel concentration.