A silver staining procedure for nucleic acids in polyacrylamide gels without fixation and pretreatment

Silver staining of nucleic acid has been widely used in molecular marker analysis such as simple sequence repeat (SSR), single-strand conformation polymorphism (SSCP), and amplified fragment-length polymorphism (AFLP). Many alternatives to silver staining methods have been described, but these methods are not efficient or cost-effective. Here we report a silver staining method that requires less than 10 min for one gel and can save chemicals as well. It has a detection limit of approximately 5.6 pg of DNA/mm² in nondenaturing polyacrylamide gels and 12.8 pg/mm² in denaturing polyacrylamide gels.     

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers^1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.     

Detection of radioactively labeled proteins is quenched by silver staining methods: Quenching is minimal for 14C and partially reversible for 3H with a photochemical stain

Silver staining methods for protein detection in polyacrylamide gels have a quenching effect on autoradiography and fluorography. This effect was quantitated for proteins in two-dimensional gels by microdensitometry using a computer equipped with an image processor and by scintillation counting of proteins solubilized from the gels. The original histologically derived silver stain had a quenching effect that was severe and irreversible for ³H detection and moderate for ¹⁴C detection. A silver stain based on photochemical methods had minimal quenching of ¹⁴C detection and less of a quenching effect than the histological stain for ³H detection. The ³H quenching effect was partially reversible for the photochemical stain.     

Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate.

The utilization of Fe²⁺-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The first procedure is used in standard disc electrophoresis and involves fixing the protein with trichloroacetic acid, staining with Fe²⁺-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate-containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe²⁺-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 μg of bovine serum albumin, and the limit of detection of this protein is 1 μg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fe²⁺-bathophenanthroline sulfonate is an excellent protein stain.     

Application of PCR-SSCP for molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust

The environmental exposure of farm children to microorganisms in dust has become a focus of interest, since microbial exposure on farms has been related to a reduced prevalence of asthma and atopic diseases in children. Previous studies almost exclusively focused on the determination of microbial counts using conventional culturing or the determination of microbial compounds i.e. endotoxins. In this study PCR-SSCP (single-strand conformation polymorphism) was modified for characterising bacterial communities in environmental dusts and their sensitivity and reproducibility was validated. A fivefold repeated PCR-SSCP analyses of a well homogenised mattress dust, cow-shed dust, swine-shed dust, chicken-shed dust and a horse-shed dust sample, respectively, showed similarities, based on Pearson correlations, ranging from 89.7% to 95.2%. The reproducibility of day to day variations (five days) and gel to gel variations (five gels) was also around 90%. The detection limit of Escherichia coli was 7 x 10(1) cfu g(-1) whereas Listeria monocytogenes and Bacillus licheniformis containing 30% spores showed visible bands at 7 x1 0(2) cfu g(-1). Application of this method to dust samples of 37 sheds and 63 children's mattresses showed that distinct farm environment dusts reflected different SSCP profiles. However, digital analysis of the gels showed that some bands in the profiles of shed- and mattress dusts were found at the same position in the gels. By excision, cloning, sequencing and phylogenetic analyses, these bands were identified as Corynebacterium tuberculostearicum, Corynebacterium mucifaciens, Staphylococcus epidermidis, Acinetobacter lwoffii, Brevibacterium iodinum, Brevibacterium linens and Arthrobacter spp, respectively. These results may reflect transfer of microorganisms from animal sheds to mattresses. In conclusion this study demonstrates that PCR-SSCP is a promising method with sensitive detection limits and moderate sample variances to be applied for epidemiological studies characterizing the exposure of farmers using environmental dust.     

Temperature dependence of the elastic properties of alginate gels

The moduli of elasticity of calcium and lead alginate gels increase with time after preparation, and the temperature dependence of the rate of syneresis suggests an activation energy of 8?12 x 10⁴ J.mol?¹ for the formation of new junctions. At zero time, a negative temperature-dependence was found for the elastic force measured at a low degree of deformation (4%). Deformation of the gels was associated with an increase in entropy and internal energy. When the calcium ions in a preformed calcium alginate gel were exchanged for lead ions, which have a higher affinity for alginate, the modulus increased due to an enhanced increase in internal energy with deformation. Reversal of the sequence of introducing the two types of ions gave the opposite effect. The data suggest that the junctions are “weak points” in the gels, and that even small deformations can cause partial rupture.     

The lens proteins in adult and embryos of the periodic albino mutant ofXenopus laevis

Summary The crystallins of normal and a^p mutants ofX. laevis have been studied using biochemical (electrophoresis in agar and polyacrylamide gels, isoelectric focusing) and immunochemical methods (immunoelectrophoresis, immunodiffusion, immunoabsorption, immunofluorescence, isoelectrofocusing with immunoidentification). The immunochemical analysis was carried out with rabbit antisera prepared against electrophoretic fractions of the mutant lens.Crystallins of adultX. laevis (a^p/a^p; ++/++) are heterogenous as judged by electrophoretic mobility, isoelectric point, antigenic and species specificity.No qualitative nor quantitative differences were found between crystallins of normal and mutant animals at the level of the protein subunits. These conclusions, however, are valid only for those crystallins, which are solubilized at pH 9.0.Immunofluorescence studies showed that crystallins appear in the normal and mutant embryos at practically the same time. No significant differences in the appearance of specific immunofluorescence between the normal and mutant embryos were found.Some of the gamma and, perhaps, beta-crystallins appear first; alpha-crystallins appear later. It has been shown for the first time that some gamma-crystallins are formed at advanced developmental stages.The periodic albino mutation does not affect the function of genes coding for crystallins either in embryos or in the adultX. laevis.     

Comparison of Crenarchaeal Consortia Inhabiting the Rhizosphere of Diverse Terrestrial Plants with Those in Bulk Soil in Native Environments

To explore whether the crenarchaeal consortium found in the rhizosphere is distinct from the assemblage of crenarchaeotes inhabiting bulk soil, PCR-single-stranded-conformation polymorphism (PCR-SSCP) profiles were generated for 76 plant samples collected from native environments. Divergent terrestrial plant groups including bryophytes (mosses), lycopods (club mosses), pteridophytes (ferns), gymnosperms (conifers), and angiosperms (seed plants) were collected for this study. Statistical analysis revealed significant differences between rhizosphere and bulk soil PCR-SSCP profiles (Hotelling paired T² test, P < 0.0001), suggesting that a distinct crenarchaeal consortium is associated with plants. In general, phylotype richness increased in the rhizosphere compared to the corresponding bulk soil, although the range of this increase was variable. Examples of a major change in rhizosphere (versus bulk soil) PCR-SSCP profiles were detected for all plant groups, suggesting that crenarchaeotes form associations with phylogenetically diverse plants in native environments. In addition, examples of minor to no detectable difference were found for all terrestrial plant groups, suggesting that crenarchaeal associations with plants are mediated by environmental conditions.     

Localization and direct quantitation of 3H-labeled proteins and RNAs in slab gels by a new detection system

The distribution patterns of ³H-labeled proteins and RNAs in dehydrated polyacrylamide and agarose gels have been recorded with a newly developed instrument: the Linear Analyzer which has a position-sensitive detector that counts a whole gel lane simultaneously. The high sensitivity makes the Linear Analyzer a suitable instrument for direct quantitation of tritium labeled macromolecules in gels without pretreatment by impregnating reagents. The very low detection limit of the Linear Analyzer reduces substantially the time of evaluation in comparison to the relativly long exposure times needed in fluorography. The resolution of radioactivity profiles monitored by the Linear Analyzer reaches the quality attained by fluorography.     

Dynamic viscoelastic properties of glycinin and β-conglycinin gels from soybeans

The effects of heating temperature on gel properties and conformational changes were investigated in glycinin and β-conglycinin gels using Theological and Fourier transform ir (FTIR) methods. Solutions of 15 wt % glycinin or β-conglycinin in 35 mM phosphate buffer at pH 7.6 were heated at various temperatures for 30 min and rheological properties were measured at 20°C. The storage modulus G′ as a function of frequency changed from a monotonical decrease with decreasing frequency to a plateau in the range from 0.0018 to 40 Hz by heating at temperatures higher than 80°C for glycinin and 65°C for β-conglycinin. A band at 1618 cm^?1 (associated with the β-sheet structure) on ir spectra increased with the formation of heat-induced gels. The value of the storage modulus G′ correlated well with the increase in absorbance at 1618 cm^?1. These results suggest that the formation of a β-sheet structure may be closely related to the value of the storage modulus G′ for heat-induced gels in soybean proteins and that heat-induced gels of glycinin and β-conglycinin are formed by cross-links with intermolecular β-sheet structures. © 1994 John Wiley & Sons, Inc.     

A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels

Electrophoresis on slab gels containing a linear gradient of polyacrylamide concentration has been used to separate DNA fragments obtained by restriction of viral DNAs. A simple method of preparing gradient gels using a sucrose density-gradient mixer and preexisting slab gel apparatus is described. DNA fragments of molecular weights 7 × 10⁴–14 × 10⁶ have been fractionated on gels of 3.5–7.5% and 2.5–7.5% acrylamide concentration. In addition to the wide range of fragment sizes which may be run on a single gel, a further advantage of the system is that much sharper bands are obtained compared to conventional constant concentration gels, thus improving resolution.In the molecular-weight range below 5 × 10⁶, for bands whose terminal velocities in the polyacrylamide concentration gradient approach zero, an approximately linear relationship holds between the logarithms of the molecular weights of the fragments and the logarithms of the distances they have migrated in the gel. Thus, by choosing a suitable upper limit to the concentration gradient, the gel system provides a method for estimating approximate molecular weights of unknown DNA fragments, by comparing their mobilities to known standards.     

Optimization of PCR protocol in microsatellite analysis with silver and SYBR® stains

Here we present the optimization of PCR conditions for microsatellite analysis of coniferous trees. The use of touchdown protocol for annealing resulted in a high success rate for optimization using fewer temperature profiles. The use of SYBR^￠ Green gel stain to detect PCR products in agarose gels was more sensitive than ethidium bromide. This is valuable for determining the success of PCR reactions and estimating the amount of PCR products formed—which is crucial in determining the dilution required to produce bands of similar intensity upon silver staining of the polyacrylamide gels. The use of SYBR^￠ Gold for staining polyacrylamide gels was not satisfactory in terms of the image quality produced. However, it was comparable to silver staining in terms of sensitivity, and could possibly be used in cases where the products are present as sharp single bands. In those cases, the use of SYBR^￠ Gold gel stain would save time and money for staining polyacrylamide gels.     

A rapid and simple method for identifying Mycobacterium tuberculosis W-Beijing strains based on detection of a unique mutation in Rv0927c by PCR-SSCP

Recently we have found that W-Beijing Mycobacterium tuberculosis strains have a unique in-frame trinucleotide (AGC) deletion at position 421 of Rv0927c and a −127G → A mutation in Rv0927c-pstS3 intergenic region. Based on detecting the 421 trinucleotide deletion of these two mutations which can alter the ssDNA conformation more extensively than the other, we developed a PCR-SSCP method for rapid identification of W-Beijing strains among non-Beijing strains. Altogether, 104 clinical isolates were analyzed, including 68 W-Beijing strains and 36 non-Beijing strains. We found that PCR-SSCP successfully differentiated all the W-Beijing strains from the non-Beijing strains. In addition, we unexpectedly discovered that SDS-PAGE protein gels had better resolving power than conventional TBE polyacrylamide gel in detecting the AGC deletion mutation in the SSCP analysis.     

Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans

doi:10.1111/j.1741‐2358.2009.00337.x
Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans Objective: The aim of this study was to examine the effectiveness of antifungal gels incorporated into a tissue conditioner which inhibits the growth of Candida albicans in vitro. Background: The release of drugs from relining materials has been demonstrated earlier. However, the incorporation of antifungal agents in gel form has not yet been studied. Materials and methods: Visco‐gel^® tissue conditioner was prepared with chlorhexidine digluconate and miconazole in gel form in a concentration of 5, 10, 15, 20 and 25% by volume. Sample discs were prepared and placed on Sabouraud Dextrose Agar (SDA) plates which had been previously inoculated with C. albicans, and incubated aerobically at 37°C. To investigate antifungal activity over time, Visco‐gel discs containing 20%v/v miconazole were prepared and immersed in water for different time periods before being placed on SDA plates inoculated with C. albicans. Results: Chlorhexidine digluconate gel added to tissue conditioner had no inhibition effect on the growth of C. albicans. Incorporation of miconazole gave a dose‐related inhibitory effect on candidal growth. Immersion of the discs in water showed an inverse relationship between time of immersion and degree of inhibition. Conclusion: Miconazole added in gel form to Visco‐gel^® had an inhibitory effect on the growth of C. albicans in vitro.     

Bradyrhizobium japonicum Survival in and Soybean Inoculation with Fluid Gels

The utilization of gels, which are used for fluid drilling of seeds, as carriers of Bradyrhizobium japonicum for soybean (Glycine max (L.) Merr.) inoculation was studied. Gels of various chemical composition (magnesium silicate, potassium acrylate-acrylamide, grafted starch, and hydroxyethyl cellulose) were used, although the hydroxyethyl cellulose gels were more extensively investigated. Gel inocula were prepared by mixing gel powder with liquid cultures of B. japonicum (2% [wt/vol]). The population of B. japonicum USDA 110 did not change in each gel type during 8 days of incubation at 28°C. These fluid gels were prepared with late-exponential-growth-phase cells that were washed and suspended in physiological saline. Mid-exponential-growth-phase B. japonicum USDA 110, 123, and 138 grew in cellulose gels prepared with yeast extract-mannitol broth as well as or better than in yeast extract-mannitol broth alone for the first 10 days at 28°C. Populations in these cellulose gels after 35 days were as large as when the gels had originally been prepared, and survival occurred for at least 70 days. Soybeans grown in sand in the greenhouse had greater nodule numbers, nodule weights, and top weights with gel inoculants compared with a peat inoculant. In soil containing 10³ indigenous B. japonicum per g of soil, inoculation resulted in increased soybean nodule numbers, nodule weights, and top weights, but only nodule numbers were greater with gel than with peat inoculation. The gel-treated seeds carried 10² to 10³ more bacteria per seed (10⁷ to 10⁸) than did the peat-treated seeds.     

Transport of probe molecules through fibrin gels as observed by means of holographic relaxation methods

Holographic relaxation spectroscopy (HRS) has been used to study transport of benzospiropyran (SP), BSA labeled with azobenzene (BSA-ABITC), and IgG-κ labeled with fluorescein (IgG-FITC) through fibrin gels formed under various conditions. The structures of the gels were controlled by means of the concentrations of fibrinogen, thrombin, and Ca²⁺ present during assembly of the fibrin. The diffusion coefficient of free dye (SP) was found to be independent of the fibrinogen concentration. The diffusion rate of labeled BSA reflected the assembly conditions of the gel for fibrinogen concentrations above approximately 6 g/L. In particular, the diffusion coefficient was higher in gels formed in the presence of 5 mM Ca²⁺. The labeled IgG showed photoinduced aggregation, as previously reported, as well as photoinduced attachment to the gel network to produce a permanent diffraction grating. Thus IgG is not a probe in the classical sense, but provides a model for protein diffusion and interactions in gels. These studies indicate that HRS is well suited to the study of molecular transport in fibrin gels.     

Direct visualization of IMP--GMP:pyrophosphate phosphoribosyltransferase in polyacrylamide gels

Direct visual detection of IMP-GMP: pyrophosphate phosphoribosyl-transferase (HGPRT) activity after separation by polyacrylamide gel electrophoresis has not been easy to achieve. Radiochemical assays have been used but they have the disadvantage of requiring considerable amounts of time before the results are available (1.2). A fluorescence assay that couples inosine 5′-monophosphate (IMP) to IMP dehydrogenase with concomitant formation of NADH has been described (3) but is inconvenient because the coupling enzyme is not commercially available. Bieber (4) has developed a fluorescence assay that can be used for HGPRT detection but it requires that the gels be sliced before they are incubated with the substrate mixture. A rapid, convenient method for visual observation of HGPRT activity would facilitate the study of this enzyme and its variants. In this paper we report the development of such a method based on precipitation of inorganic pyrophosphate, one of the reaction products, with Mn²⁺.     

Solution NMR of proteins within polyacrylamide gels: Diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes

The diffusive properties of biomacromolecules within the aqueous phase of polyacrylamide gels are described. High quality NMR spectra can be obtained under such conditions. As compared to water, a fivefold reduction in the translational diffusion constant, but only a 1.6-fold decrease (1.4-fold increase) in amide-¹⁵N T₂ (T₁) are observed for human ubiquitin within a 10% acrylamide gel. Weak alignment of the solute macromolecules can be achieved within such gels by vertical or radial compression or by the embedding of magnetically oriented purple membrane fragments. The methods are applied to derive residual dipolar couplings for human HIV-1 Nef and ubiquitin.     

Curdlan gels as protein drug delivery vehicles

The use of curdlan, a natural -1,3-glucan, in protein drug delivery vehicles was studied by carrying out in vitro release studies with curdlan gels containing bovine serum albumin (BSA) as a model protein. Addition of urea (8 M) decreased the gel formation temperature to 37°C. Curdlan was hydroxyethylated in order to form gels under mild conditions such as physiological temperature and pH. In gels formed in 8 M urea solution, urea was almost released after 2 h while BSA was completely released after 45–100 h. The total time for complete release of BSA increased with curdlan concentration within gels. The strength of hydroxyethylated curdlan gels (385.7 dyne cm^–2) was weaker than that of curdlan gels formed in 8 M urea solution (6277 dyne cm^–2).     

Regulation of gene expression in Dictyostelium discoideum cells exposed to immobilized carbohydrates

When amoebae of Dictyostelium discoideum develop on gels of polyacrylamide that are derivatized with glucosides, they become capable of aggregation at the same time as cells not exposed to glucosides. However, the aggregation centers and streams of adherent cells formed on immobilized glucosides suddenly disintegrate. The cells repeatedly re-aggregate, but never form tight aggregates as they do on other substrata. Tight aggregates formed in the absence of glucosides disperse after their transfer to glucoside gels, and the cells undergo aggregation-disaggregation cycles. The formation of tight aggregates is correlated with the expression of specific post-aggregative poly(A)⁺ RNAs. These RNAs are not expressed in cells developing on glucoside gels, and the dispersal of tight aggregates on such gels is accompanied by the almost complete loss of these RNAs. A developmentally regulated membrane glycoprotein called contact site A, which is a marker of aggregation-competent cells, is normally expressed on glucoside gels. Cyclic AMP is also produced, indicating that the strong increase of adenylate cyclase activity during the preaggregation phase is not affected. In conclusion, cell contact with immobilized glucosides specifically inhibits postaggregative gene expression and arrests development at the aggregation stage.