Fluorescent microplate-based analysis of protein-DNA interactions II:immobilized DNA

A simple protein-DNA interaction analysis has been developed using both a high-affinity/high-specificity zinc finger protein and a low-specificity zinc finger protein with nonspecific DNA binding capability. The latter protein is designed to mimic background binding by proteins generated in randomized or shuffled gene libraries. In essence, DNA is immobilized onto the surface of microplate wells via streptavidin capture, and green fluorescent protein (GFP)-labeled protein is added in solution as part of a crude cell lysate or protein mixture. After incubation and washing, bound protein is detected in a standard microplate reader. The minimum sensitivity of the assay is approximately 0.4 nM protein. The assay format is ideally suited to investigate the interactions of DNA binding proteins from within crude cell extracts and/or mixtures of proteins that may be encountered in protein libraries generated by codon randomization or gene shuffling.

Statnote 10: the two-way analysis of variance

The two-way design has been variously described as a matched-sample F-test, a simple within-subjects ANOVA, a one-way within-groups ANOVA, a simple correlated-groups ANOVA, and a one-factor repeated measures design! This confusion of terminology is likely to lead to problems in correctly identifying this analysis within commercially available software. The essential feature of the design is that each treatment is allocated by randomization to one experimental unit within each group or block. The block may be a plot of land, a single occasion in which the experiment was performed, or a human subject. The ‘blocking’ is designed to remove an aspect of the error variation and increase the ‘power’ of the experiment. If there is no significant source of variation associated with the ‘blocking’ then there is a disadvantage to the two-way design because there is a reduction in the DF of the error term compared with a fully randomised design thus reducing the ‘power’ of the analysis.

Raman microscopy of formamide-intercalated kaolinites treated by controlled-rate thermal analysis technology

Raman spectroscopy of formamide-intercalated kaolinites treated using controlled-rate thermal analysis technology (CRTA), allowing the separation of adsorbed formamide from intercalated formamide in formamide-intercalated kaolinites, is reported. The Raman spectra of the CRTA-treated formamide-intercalated kaolinites are significantly different from those of the intercalated kaolinites, which display a combination of both intercalated and adsorbed formamide. An intense band is observed at 3629 cm-1, attributed to the inner surface hydroxyls hydrogen bonded to the formamide. Broad bands are observed at 3600 and 3639 cm-1, assigned to the inner surface hydroxyls, which are hydrogen bonded to the adsorbed water molecules. The hydroxyl-stretching band of the inner hydroxyl is observed at 3621 cm-1 in the Raman spectra of the CRTA-treated formamide-intercalated kaolinites. The results of thermal analysis show that the amount of intercalated formamide between the kaolinite layers is independent of the presence of water. Significant differences are observed in the CO stretching region between the adsorbed and intercalated formamide.

Detailed analysis of a conservative difference approximation for the time fractional diffusion equation

Diffusion equations that use time fractional derivatives are attractive because they describe a wealth of problems involving non-Markovian Random walks. The time fractional diffusion equation (TFDE) is obtained from the standard diffusion equation by replacing the first-order time derivative with a fractional derivative of order α ∈ (0, 1). Developing numerical methods for solving fractional partial differential equations is a new research field and the theoretical analysis of the numerical methods associated with them is not fully developed. In this paper an explicit conservative difference approximation (ECDA) for TFDE is proposed. We give a detailed analysis for this ECDA and generate discrete models of random walk suitable for simulating random variables whose spatial probability density evolves in time according to this fractional diffusion equation. The stability and convergence of the ECDA for TFDE in a bounded domain are discussed. Finally, some numerical examples are presented to show the application of the present technique.

Determination of preventive maintenance lead time using hybrid analysis

The time for conducting Preventive Maintenance (PM) on an asset is often determined using a predefined alarm limit based on trends of a hazard function. In this paper, the authors propose using both hazard and reliability functions to improve the accuracy of the prediction particularly when the failure characteristic of the asset whole life is modelled using different failure distributions for the different stages of the life of the asset. The proposed method is validated using simulations and case studies.

Hydrazine-hydrate intercalated halloysite under controlled-rate thermal analysis conditions

The thermal behaviour of halloysite fully expanded with hydrazine-hydrate has been investigated in nitrogen atmosphere under dynamic heating and at a constant, pre-set decomposition rate of 0.15 mg min-1. Under controlled-rate thermal analysis (CRTA) conditions it was possible to resolve the closely overlapping decomposition stages and to distinguish between adsorbed and bonded reagent. Three types of bonded reagent could be identified. The loosely bonded reagent amounting to 0.20 mol hydrazine-hydrate per mol inner surface hydroxyl is connected to the internal and external surfaces of the expanded mineral and is present as a space filler between the sheets of the delaminated mineral. The strongly bonded (intercalated) hydrazine-hydrate is connected to the kaolinite inner surface OH groups by the formation of hydrogen bonds. Based on the thermoanalytical results two different types of bonded reagent could be distinguished in the complex. Type 1 reagent (approx. 0.06 mol hydrazine-hydrate/mol inner surface OH) is liberated between 77 and 103°C. Type 2 reagent is lost between 103 and 227°C, corresponding to a quantity of 0.36 mol hydrazine/mol inner surface OH. When heating the complex to 77°C under CRTA conditions a new reflection appears in the XRD pattern with a d-value of 9.6 Å, in addition to the 10.2 Ĺ reflection. This new reflection disappears in contact with moist air and the complex re-expands to the original d-value of 10.2 Å in a few h. The appearance of the 9.6 Å reflection is interpreted as the expansion of kaolinite with hydrazine alone, while the 10.2 Å one is due to expansion with hydrazine-hydrate. FTIR (DRIFT) spectroscopic results showed that the treated mineral after intercalation/deintercalation and heat treatment to 300°C is slightly more ordered than the original (untreated) clay.