Impact of Quenching Failure of Cy Dyes in Differential Gel Electrophoresis

Background: Differential gel electrophoresis (DIGE) is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. Methodology/Principal Findings: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. Conclusions/Significance: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration

Fine Root Productivity and Dynamics on a Forested Floodplain in South Carolina

The highly dynamic, fine-root component of forested wetland ecosystems has received inadequate attention in the literature. Characterizing fine root dynamics is a challenging endeavor in any system, but the difficulties are particularly evident in forested floodplains where frequent hydrologic fluctuations directly influence fine root dynamics. Fine root (\u3c 3mm) biomass, production, and turnover were estimated for three soils exhibiting different drainage patterns within a mixed-oak community on the Coosawhatchie River floodplain, Jasper County, SC. Within a 45-cm deep vertical profile, 74% of total fine root biomass was restricted to the upper 15 cm of the soil surface. Fine root biomass decreased as the soil became less well-drained (e.g., fine root biomass in well-drained soil \u3e intermediately drained soil \u3e poorly drained soil). Fine root productivity was measured for one year using minirhizotrons and in-situ screens. Both methods suggested higher fine root production in better drained soils but showed frequent fluctuations in fine root growth and mortality, suggesting the need for frequent sampling at short intervals (e.g., monthly) to accurately assess fine root growth and turnover. Fine root production, estimated with in-situ screens, was 1.5, 1.8, and 0.9 Mg ha-1 yr-1 in the well-drained, intermediately drained, and poorly drained soils, respectively. Results from minirhizotrons indicated that fine roots in well-drained soils grew to greater depths while fine roots in poorly drained soils were restricted to surface soils. Minirhizotrons also revealed that the distribution of fine roots among morphological classes changed between well-drained and poorly drained soils

Nitrogen to phosphorus ratio of plant biomass versus soil solution in a tropical pioneer tree, Ficus insipida

It is commonly assumed that the nitrogen to phosphorus (N:P) ratio of a terrestrial plant reflects the relative availability of N and P in the soil in which the plant grows. Here, this was assessed for a tropical pioneer tree, Ficus insipida. Seedlings were grown in sand and irrigated with nutrient solutions containing N:P ratios ranging from <1 to >100. The experimental design further allowed investigation of physiological responses to N and P availability. Homeostatic control over N:P ratios was stronger in leaves than in stems or roots, suggesting that N:P ratios of stems and roots are more sensitive indicators of the relative availability of N and P at a site than N:P ratios of leaves. The leaf N:P ratio at which the largest plant dry mass and highest photosynthetic rates were achieved was ∼11, whereas the corresponding whole-plant N:P ratio was ∼6. Plant P concentration varied as a function of transpiration rate at constant nutrient solution P concentration, possibly due to transpiration-induced variation in the mass flow of P to root surfaces. The transpiration rate varied in response to nutrient solution N concentration, but not to nutrient solution P concentration, demonstrating nutritional control over transpiration by N but not P. Water-use efficiency varied as a function of N availability, but not as a function of P availability

Controlling gold nanoparticle assembly on electron beam-reduced nitrophenyl self-assembled monolayers <i>via</i> electron dose

Electron beam lithography is a well-established tool suitable for the modification of substrate surface chemistry. It therefore follows that the deposition and self-assembly of nanoparticles on a surface can be directed using this method. This work explores the effect of electron dose on the electron beam lithographic patterning of self-assembled monolayers (SAMs) on gold surfaces. Electron beam irradiation of the sample induces conversion of the SAM terminal functional aromatic nitro (NO2) moieties to aromatic amino (NH2) moieties. The cationic NH2 functionalised regions direct the site-specific assembly of anionic citrate-passivated gold nanoparticles in aqueous solution at pH 4.5. Control of nanoparticle attachment to the SAM is demonstrated over the exposure range 5000–125,000 uC/cm2. Overexposure led to significant numbers of secondary electrons reaching the surface, causing conversion of functional aromatic moieties outside of the regions irradiated, which reduced feature quality and regional selectivity of adsorption