Preparative Isoelectric Focusing

Electrophoretic mobility has been used for years as a basis for separation and characterization of proteins. The resolving power of conventional electrophoresis is limited, and the characterization by mobility is not unique because of the dependence of the protein mobilities on the pH and ionic strength of the buffers.     

Melting temperature evolution of non-reorganized crystals. Poly(3-hydroxybutyrate)

In the present study the correlation between the melting behaviour of poly(3-hydroxybutyrate) (PHB) original, non-reorganized crystals and the crystallinity increase during isothermal crystallization is presented and discussed. Since the reorganization processes modify the melting curve of original crystals, it is necessary to prevent and hinder all the processes that influence and increase the lamellar thickness. PHB exhibits melting/recrystallization on heating, the occurring of lamellar thickening in the solid state being excluded. The first step of the study was the identification of the scanning rate which inhibits PHB recrystallization at sufficiently high T_c. For the extrapolated onset and peak temperatures of the main melting endotherm, which is connected to fusion of dominant lamellae, a double dependence on the crystallization time was found. The crystallization time at which T_onset and T_peak change their trends was found to correspond to the spherulite impingement time, so that the two different dependencies were put in relation with primary and secondary crystallizations respectively. The increase of both T_onset and T_peak at high crystallization times after spherulite impingement was considered an effect due to crystal superheating and an indication of a stabilization process of the crystalline phase. Such stabilization, which produces an increase of the melting temperature, is probably connected with the volume filling that occurs after spherulite impingement.     

Dynamic switching between binding sites in the complexation of macrocyclic ‘push-pull’ chromophores to lanthanides

The introduction of a 1,3-diacetylpyridine moiety, as an additional binding niche in a macrocyclic receptor ligand containing a conjugated, push-pull malonate functionality, leads to, in addition to the 1:1 binding stoichiometry, the equilibrium formation of multiple complexes, specifically 1:2, 2:1, and 3:1 ligand:metal complexes with lanthanide trications. Various binding modes elicit distinctive responses in the visible region, which enhance the potential recognition of lanthanide ions. This paper also highlights the usefulness of a full factor analysis in the elucidation of complex binding phenomena.     

Efficient removal of DNA from proteomic samples prior to two-dimensional map analysis

Several methods have been described in the literature for removal of DNA from protein samples prior to proteome analysis. They in general involve protein precipitation techniques. In other protocols, DNAse treatment is suggested prior to precipitation of proteins in excess acetone. All these methods have been evaluated and found to perform poorly in DNA removal, as illustrated by two-dimensional (2D) maps where horizontal and vertical sample streaking are still substantial. Such removal is in general necessary in tissue lysates and especially when analysing sub-cellular organelles, such as nuclei, where the high DNA levels strongly interfere with proteome analysis. Another method is proposed here for efficient DNA removal: two-phase extraction of DNA in chloroform/phenol/isoamyl alcohol, a procedure commonly used to rid DNA samples of protein contaminants, but rarely applied to protein preparation. This extraction is not very efficient if performed at slightly acidic to neutral pH values, but it performs extremely well at pH values of 9.5 or higher. The 2D maps thus obtained of Escherichia coli lysates as well as extracts from purified nuclei of eukaryotic cells are not only devoid of any vertical or horizontal streaking, but exhibit many more spots, especially in the alkaline region of the 2D gels, suggesting that these basic proteins were in general lost to proteome analysis due to co-precipitation in tenacious protein–DNA complexes. It is hypothesized that the alkaline pH values adopted in the two-phase extraction help to fully disrupt any residual DNA–protein complexes, due to strong Coulombic repulsion.     

Immobilized pH gradients (IPG) simulator--an additional step in pH gradient engineering: II. Nonlinear pH gradients

While in the companion paper (Tonani, C. & Righetti, P. G., Electrophoresis 1991, 12, 1011-1021) we gave the general outline of our new computer program, immobilized pH gradients (IPG) simulator, able to simulate and optimize linear pH gradients for isoelectric focusing in immobilized pH gradients, in the present report we extend the application of such a program to: (i) convex exponential gradients, (ii) logarithmic and (iii) polynomial gradients. Such gradients are meant to give equal space to protein spots in complex protein mixtures (e.g., cell lysates, biological fluids) and follow the statistical distribution of protein pI values along the pH axis. They will prove of fundamental importance in two-dimensional maps, both because they optimize the spreading of spots in the two-dimensional plane and because of the excellent reproducibility of immobilized pH gradients. The following concave exponential recipes are given: pH 3-8, pH 3-9, pH 3-10, pH 3-11, pH 4-7, pH 4-8, pH 4-9, pH 4-10, pH 4-11, pH 5-8, pH 5-9, and pH 5-10, as well as the most extended pH 2.5-11 interval. Two interesting logarithmic gradients are described: pH 3-6 and pH 3-7 and one sigmoidal (derived with a polynomial of 5th degree): pH 3-11.     

An image system and surface singularity solutions for potential flow past an elliptical cylinder

The Milne-Thomson circle theorem is extended to give a simplegeneral expression for the image system in an elliptical cylinderintroduced into an otherwise specified unbounded potential flowwhich contains no singularities in the region to be occupiedby the ellipse. This image system is used to obtain an expressionfor the corresponding source-sink surface singularity distributionon the ellipse, thus providing new benchmark test cases forsource-sink solutions as obtained numerically by a panel method.Several typical examples are given to illustrate the generaltheoretical approach.