Diffusion-induced spontaneous pattern formation on gelation surfaces

Although the pattern formation on polymer gels has been considered as a result of the mechanical instability due to the volume phase transition, we found a macroscopic surface pattern formation not caused by the mechanical instability. It develops on gelation surfaces, and we consider the reaction-diffusion dynamics mainly induces a surface instability during polymerization. Random and straight stripe patterns were observed, depending on gelation conditions. We found the scaling relation between the characteristic wavelength and the gelation time. This scaling is consistent with the reaction-diffusion dynamics and would be a first step to reveal the gelation pattern formation dynamics.Comment: 7 pages, 4 figure

Analysis and characterization of heparin impurities

This review discusses recent developments in analytical methods available for the sensitive separation, detection and structural characterization of heparin contaminants. The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007–2008 spawned a global crisis resulting in extensive revisions to the pharmacopeia monographs on heparin and prompting the FDA to recommend the development of additional physicochemical methods for the analysis of heparin purity. The analytical chemistry community quickly responded to this challenge, developing a wide variety of innovative approaches, several of which are reported in this special issue. This review provides an overview of methods of heparin isolation and digestion, discusses known heparin contaminants, including OSCS, and summarizes recent publications on heparin impurity analysis using sensors, near-IR, Raman, and NMR spectroscopy, as well as electrophoretic and chromatographic separations

Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining

Discontinuous buffer system for polyacrylamide and agarose gel electrophoresis of DNA fragments

DNA fragments up to 9 kb in size were stacked and separated by polyacrylamid gel electrophoresis, and those up to 50 kb in size by agarose gel electrophoresis using a discontinuous buffer system. Polyacrylamide gel at pH 8.9, 2°C, 0.01 M ionic strength, yielded sharp bands with DNA loads of 8 μg/cm2 of gel of a mixture of 19 DNA fragments in the size range of 72–23130 bp, while agarose gels at pH 8.5, 25°C, provided well-resolved, unperturbed bands at 0.04 M ionic strength with DNA loads of 1 μg/cm2 of the same mixture. Note that the ionic strength of the agarose gels is comparable to the conventionally used 0.5 × TBE (Tris-borate-EDTA) buffer, while that successfully applied to polyacrylamide is seven-fold less than the ionic strength of conventionally used 1 × TBE buffer, with is substantially shorter duration of electrophoresis as a result. The application of a discontinuous buffer system to the gel electrophoresis of DNA results in (i) Band identification Rf, the migration distance relative to a sharply defined “buffer front” (moving boundary). This is sufficiently labor saving, compared to determining absolute mobilities, so as to render practical the expression of bands as numbers, with benefits for data storage, statistical manipulations and physico-chemical exploitation of mobility data. The use of Rf's also circumvents loss of precision in mobility measurement resulting from progressive band spreading of dye bands used as a front. (ii) A uniformly and highly concentrated starting zone, beneficial to resolution, is obtained, without the losses by which separate concentration steps are usually burdened. (iii) The degree of dilution of the DNA sample becomes unimportant

Information on DNA conformation derived from the Ferguson plot of DNA fragments of up to 9 kb in size, using polyacrylamide gel electrophoresis in a discontinuous buffer system

The Ferguson plot in polyacrylamide gel electrophoresis (PAGE) (15 %CDATD, moving boundary electrophoresis buffer system operative at pH 8.9, 4°C, 8 mA/cm2 of gel) of DNA fragments up to 9.4 kb in size was found to exhibit a linear segment at polyacrylamide concentrations starting at 3 %T and undergoing a gradual transition into a concave segment at higher gel concentrations, confirming previous findings by Stellwagen [1]. The larger the DNA, and the higher the gel concentration, the less extended the linear and the more extended the concave segment of the plot. The lowest %T of the linear range for DNA in polyacrylamide remains unknown since mobilities at nongelling concentrations below 3%T have not as yet been measured. As previously suggested [1, 2], the transition from the linear to the concave segment corresponds to that from the randomly oriented DNA to the anisotropically stretched “reptating” DNA. For a DNA of 9.4 kb in size, the end of the linear range of the Ferguson plot can be extended from 3.5 to 5 %T when 15 % DATD rather than 2.5 % Bis is used to crosslink the polyacrylamide. Increasing the temperature of PAGE from 4°C to 25 and 50°C widens the linear segment progressively, indicating an increasingly random orientation with rising temperature. When current density is increased from 8 to 40 mA/cm2, the concave curvature of the Ferguson plot of DNA 1 to 9.4 kb in size decreases, suggesting a transition from a “reptating” to a randomly distributed molecule, due to increased Joule heat. Increasing the ionic strength from 0.01 to 0.04 M similarly appears to lead to a relative linearization of the Ferguson plot, suggesting a similar effect of Joule heat. Prestaining of DNA with ethidium bromide results in a decrease of both its size and its free mobility. This effect is exacerbated as the size of DNA increases from 0.1 to 9.4 kb

Physical identification of a virus in a crude leaf extract by its ferguson plot in agarose gel electrophoresis

Crude extracts of turnip crinkle virus upon agarose gel electrophoresis yield (i) virus patterns unperturbed by contaminants; (ii) plots of mobility vs. gel concentration (Ferguson plots) parallel with those of the purified virus. The parallelism suggests similarity in size and shape but a lower net charge for the crude virus. This result is obtained when gel electrophoresis is carried out either in a continuous buffer or in a discontinuous (moving boundary electrophoresis) buffer system. The latter mode has the substantial benefit of electrophoretic (auto-)concentration of dilute virus sample prior to resolution. Thus, the Ferguson plot analysis in a discontinuous buffer system of turnip crinkle virus can be viewed as a model procedure for the physical identification of other viruses contained in dilute extracts, feasible even in the absence of a prior knowledge as to the nature of, or isolation of, the virus

Agarose electrophoresis of DNA in discontinuous buffers, using a horizontal slab apparatus and a buffer system with improved properties

Using a horizontal slab apparatus with a buffer in the reservoirs at the level of the gel (“sea-level electrophoresis”), the retrograde discontinuous buffer system reported by Wiltfang et al. for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of proteins was applied to DNA electrophoresis. This application yielded the advantages of an increased displacement rate of the moving boundary front and a decrease in the concentration of the counterion base in the resolving phase, which yielded reduced relative mobility values at equivalent gel concentrations and practicable low buffer concentrations. The change of relative mobilities (Rf) with a variation of field strength is decreased compared to that of the migration rate in the continuous Tris-boric-acid-EDTA (TBE) buffer and thus the robustness of the system is improved, as well as the efficiency of separation. The system of Wiltfang et al. has in common with previously described discontinuous DNA systems, that it is able to stack DNA from dilute samples and is insensitive to sample components with lower net mobilities than DNA, such as acetate. However, the variance of Rf at constant current density in the discontinuous buffer system is not improved over that of the migration rate at constant field strength in the continuous TBE buffer