Ternary complexes in agarose gels prepared from binary solvents

The thermoreversible gelation of agarose has been investigated in four different aqueous binary solvents: Water/dimethyl sulfoxide, water/N,N-dimethylformamide, water/N-methylformamide, and water/formamide. The phase diagrams have been subsequently established as a function of agarose concentration and solvent composition. These diagrams suggest the formation of ternary complexes agarose/water/cosolvent.     

Oxygen-17 relaxation in aqueous agarose gels

Nuclear magnetic relaxation of oxygen-17 in H₂¹⁷O enriched agarose gels shows that existing explanations of water behaviour are oversimplified. Satisfactory models must include at least three proton phases, two of which involve water molecules.     

Enhancing the sensitivity of DNA detection and recovery from agarose gels

By inserting nitrocellulose strips into agarose gels alongside the electrophoresed lanes and passing an electric current perpendicularly in the direction of the strips, highly efficient transfer of DNA bands onto the membrane in the form of concentrated dots is achieved. DNA detection limits by this technique are enhanced, at least three times as visualized by ethidium bromide fluorescence and at least twice more by radiolabeling.     

Internal water molecules and magnetic relaxation in agarose gels

Agarose gels have long been known to produce exceptionally large enhancements of the water 1H and 2H magnetic relaxation rates. The molecular basis for this effect has not been clearly established, despite its potential importance for a wide range of applications of agarose gels, including their use as biological tissue models in magnetic resonance imaging. To resolve this issue, we have measured the 2H magnetic relaxation dispersion profile from agarose gels over more than 4 frequency decades. We find a very large dispersion, which, at neutral pH, is produced entirely by internal water molecules, exchanging with bulk water on the time scale 10(-8)-10(-6) s. The most long-lived of these dominate the dispersion and give rise to a temperature maximum in the low-frequency relaxation rate. At acidic pH, there is also a low-frequency contribution from hydroxyl deuterons exchanging on a time scale of 10(-4) s. Our analysis of the dispersion profiles is based on a nonperturbative relaxation theory that remains valid outside the conventional motional-narrowing regime. The results of this analysis suggest that the internal water molecules responsible for the dispersion are located in the central cavity of the agarose double helix, as previously proposed on the basis of fiber diffraction data. The magnetic relaxation mechanism invoked here, where spin relaxation is induced directly by molecular exchange, also provides a molecular basis for understanding the water 1H relaxation behavior that governs the intrinsic magnetic resonance image contrast in biological tissue.     

Direct chemiluminescent immunodetection of proteins in agarose gels

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.     

Well-defined star-shaped calcite crystals formed in agarose gels

Single crystals of calcite exhibiting a morphology of well-defined 8-armed stars, which evolved from original rhombohedral calcite crystals with their 8 points extending radially into eight arms, were produced by crystallization of CaCO3 in agarose gels.     

Electrotitration curves of human gastric pepsinogens in agarose gels

Electrotitration curves (ETC) of a marker protein mixture, pH 2.5-5.65, and human pepsinogens were performed in an agarose gel, containing 2% acid carrier ampholytes, forming a pH range of 2.5-5. Although the establishment of the pH gradient by isoelectric focusing was not quite complete and linear, both biochemically and immunochemically different types of pepsinogen C (PGC) and pepsinogen A (PGA) zymogens as well as the acid isoelectric points (pI) marker proteins were separated with good resolution. Three main fractions of PGA (Pg3, Pg4, and Pg5) were detected. To obtain an exact determination of the pepsinogen pIs, a simple and very fast 10 s pressure blot technique was applied. Human pepsinogens were separated alone or mixed with pI marker proteins in the pH range 2.4-5.65. No effect of the markers was observed on the pepsinogen migration. To visualize the different protein samples in the gel and on nitrocellulose membrane, we have used colloidal gold (AuroDye) staining, proteolytic activity, and immunostaining with monoclonal antibodies anti PGA and PGC. The described method shows an ability to separate proteins at acidic conditions with a resolution comparable to isoelectric focusing with immobilized pH gradients, but much faster, easier, and cheaper. In addition, the technique allows us to determine precise and exact pI values, and is suitable for studies of the pepsinogen polymorphism and its role in gastric diseases.     

A method for recovery of native, clonally-restricted immunoglobulins from agarose gels

Multiple low level, clonally-restricted, immunoglobulins (Ig) are commonly encountered on routine serum protein electrophoresis by clinical laboratories using high resolution zone electrophoresis on agarose. We sought a method for recovering the clonally-restricted Ig, in native configuration, from clinical laboratory gels as a first step in the investigation of its clinical significance. We found that a two-stage electrophoretic procedure gave consistently good recoveries. After routine agarose gel electrophoresis, portions of the electropherogram, containing clonally-restricted Ig, were excised and subjected to flatbed isoelectric focusing in agarose to enhance separation of the individual antibody clonotypes. Multiple slabs, containing the same clonally-restricted Ig, could be cut from adjacent tracks (i.e., tracks loaded with the same specimen) on the zone electropherogram and applied to a single track on the focusing gel to improve separation and increase yields. The focused gels were cut to isolate slabs containing individual clonotypes. These slabs were washed to remove carrier ampholytes and held at -20 degrees C overnight. Ig was extracted from the thawed gels, with 61-68% recovery, by ultracentrifugation following physical disruption of the gel. Antigen binding activity of the recovered Ig was verified by rate nephelometry. Clonally-restricted antibodies were successfully isolated from an immune animal serum by this procedure and biotinylated for use as probes on Western blots.     

Diffusion of sodium dodecyl sulfate micelles in agarose gels

The gradient diffusion of ionic sodium dodecyl sulfate micelles in agarose gel was investigated at moderate concentrations above the CMC. Of particular interest were the effects of micelle, gel, and sodium chloride concentration on the micelle diffusivity. Holographic interferometry was used to measure the gradient diffusion coefficient at three sodium chloride concentrations (0, 0.03, 0.10 M), three gel concentrations (0, 1, 2 wt%), and several surfactant concentrations. Time-resolved fluorescence quenching was used to measure aggregation numbers both in solution and gel. The micelle diffusivity increased linearly with surfactant concentration at the two larger sodium chloride concentrations and all gel concentrations. In general, the strength of this effect increased with decreasing sodium chloride concentration and increased with gel concentration. This behavior is evidence of decreasing micelle-micelle electrostatic interactions with increasing sodium chloride concentrations, and increasing excluded volume effects and hydrodynamic screening with increasing gel concentration, respectively. The only exception was at 0.1M sodium chloride and 2 wt% agarose, which showed a slight reduction in the slope compared to 1 wt% agarose. It was found that the concentration effect is quite strong for charged solutes: at a NaCl concentration of 0.03 M in a 2% agarose gel, in a solution with 3% SDS micelles by volume, the micelle diffusion coefficient is doubled relative to its value in the same gel at infinite dilution. The extrapolated, infinite-dilution diffusion coefficients and the rate at which the micelle diffusivity increased with surfactant concentration were compared with predictions of previously published theories in which the micelles are treated as charged, colloidal spheres and the gel as a Brinkman medium. The experimental data and theoretical predictions were in good agreement.     

Organic light‐emitting devices having chemically‐doped arylamine oligomer as a hole injection layer

A soluble and thermally stable arylamine oligomer containing difluorenyl groups was prepared and applied to organic light‐emitting devices (OLEDs) as a hole injection layer. The oligomer layer was doped with a Lewis acid and formed by spin coating from the dichloroethane solution. The OLED with a structure of indium tin oxide (ITO)/Lewis‐acid‐doped arylamine oligomer/N,N′‐dinaphthyl‐N,N′‐diphenyl bendizine (α‐NPD)/tris(8‐quinolinolato)aluminum(III) (Alq₃)/LiF/Al showed lower drive voltages and higher power efficiencies, compared with the devices without the hole injection oligomer layer. Copyright © 2005 John Wiley & Sons, Ltd.     

Ternary complexes in analytical chemistry

Reactions between a complex AB and a third component C do not always proceed by a displacement mechanism governed by the energy difference of the chemical bonds A-B and A-C. The third component often becomes part of the complex, forming a mixed co-ordination sphere or ternary complex. The properties of this ternary complex ABC are not additive functions of the properties of AB and AC. Such reactions are important in many methods in analytical chemistry, particularly in photometric analysis, extractive separation, masking, etc. The general properties of the four basic types of ternary complex are reviewed and examples given. The four types comprise the systems (a) metal ion, electronegative ligand, organic base, (b) one metal ion, two different electronegative ligands, (c) ternary heteropoly acids, and (d) two different metal ions, one ligand.     

Stabilizing labile DNA–protein complexes in polyacrylamide gels

The electrophoretic mobility‐shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA–protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K_a>10⁹ M^?1 under normal conditions of salt and pH. Many DNA–protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein–DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.     

New hydrogels based on the interpenetration of physical gels of agarose and chemical gels of polyacrylamide

In this paper we report a novel method for preparing interpenetrating polymer hydrogels of agarose and polyacrylamide (PAAm) in three steps. The procedure consists in (i) formation of physical hydrogels of agarose, (ii) diffusion of acrylamide, N,N′-methylene-bis-acrylamide and potassium persulfate (the initiator) from aqueous solutions inside the gel of agarose, and (iii) cross-linking copolymerization reaction of the aforementioned reactants to produce PAAm chemical gels interpenetrated with the agarose physical gels. Viscoelasticity measurements and thermal analysis have been performed in order to follow the kinetics of copolymerization. The viscoelastic, swelling and thermal properties of the resulting hydrogels confirm the formation of an interpenetrated system. Further evidence of interpenetration is obtained from inspection with atomic force microscopy. The improvement of the agarose and PAAm gel properties in the resulting interpenetrated hydrogel is analyzed in view of the results.     

Self‐ and cross‐associations in two‐component mixed polymer gels

The gel properties of two‐component mixed polymer gels are investigated using a cascade model, which assumes that the gel network is formed via the self‐association of one of the two components and the cross‐association of the two components. The effects of the model parameters, such as the equilibrium constants and the functionalities for cross‐associations and self‐associations, on the composition dependence of the modulus and gel point curves are examined to elucidate the contribution of self‐associations to the gel network. The results show that the characteristics of self‐associations become pronounced when the equilibrium constant or the functionality for self‐associations is comparable to that for cross‐associations. The model is applied to analyze the critical gelling concentration data for xanthan/locust bean gum mixed gels, which shows significant self‐associations at high xanthan compositions. The resulting model curves agree well with the experimental data at all temperatures. The analysis of the temperature dependence of the best‐fit equilibrium constant yields values of enthalpy change that are consistent with previous findings. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 80–91, 2008     

Temperature‐sensitive polyzwitterionic gels

An abrupt change in the polyzwitterionic swelling ratio as a function of the temperature, sodium chloride concentration and chemical crosslinking density is established. These results are reasonably explained by the model presuming zwitterionic cluster formation in the segments between the chemical junction points. The zwitter‐ionic clusters, produced at low chemical crosslinking density only, act as temperature‐ and salt concentration‐dependent physical junction points. An irreversible temperature‐stimulated swelling‐shrinking behaviour of the polyzwitter‐ionic hydrogels (swelling ratio‐temperature hysteresis loop) at low chemical cross‐linking density is also observed and analysed.     

Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels

The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.     

SAXS study on complexes formed by anionic poly(sodium methacrylate‐co‐N‐isopropylacrylamide) gels with cationic surfactants

Small‐angle X‐ray scattering (SAXS) has been used to study the nanostructures of complexes formed by slightly crosslinked anionic copolymer gels of poly(sodium methacrylate‐co‐N‐isopropylacrylamide) [P(MAA/NIPAM)] interacting with cetylpyridinium bromide (CPB), and alkyltrimethylammonium bromide (C_nTAB, 10 ≤n ≤ 18), respectively. Both the charge density of polyelectrolyte gels and the surfactant alkyl tail length could induce the phase structure transition from Pm3n space group cubic to hexagonal close packing of spheres (HCP), while the different polar groups of pyridinium and trimethylammonium with the same hydrophobic cetyl chain in surfactants had no significant effects on the structures of complexes formed with the same gels. The highly ordered structures were shown to be formed by the self‐assembly of ionic surfactants inside the anionic gel network, driven by both electrostatic and hydrophobic interactions. Freeze drying the water‐equilibrated complexes could collapse the formed ordered structures. However, the highly ordered structures could be restored after the dried complexes were reswollen by water under the same conditions, indicating that the highly ordered water‐equilibrated complexes were thermodynamically stable. Copyright © 2000 John Wiley & Sons, Ltd.