Comparative fluorescence two-dimensional gel electrophoresis using a gel strip sandwich assembly for the simultaneous on-gel generation of a reference protein spot grid

The comparison of proteins separated on 2DE is difficult due to gel-to-gel variability. Here, a method named comparative fluorescence gel electrophoresis (CoFGE) is presented, which allows the generation of an artificial protein grid in parallel to the separation of an analytical sample on the same gel. Different fluorescent stains are used to distinguish sample and marker on the gel. The technology combines elements of 1DE and 2DE. Special gel combs with V-shaped wells are placed in a stacking gel above the pI strip. Proteins separated on the pI strip are electrophoresed at the same time as marker proteins (commercially available purified protein of different molecular weight) placed in V-wells. In that way, grids providing approximately 100 nodes as landmarks for the determination of protein spot coordinates are generated. Data analysis is possible with commercial 2DE software capable of warping. The method improves comparability of 2DE protein gels, because they are generated in combination with regular in-gel anchor points formed by protein standards. This was shown here for two comparative experiments with three gels each using Escherichia coli lysate. For a set of 47 well-defined samples spots, the deviation of the coordinates was improved from 7% to less than 1% applying warping using the marker grid. Conclusively, as long as the same protein markers, the same size of pI-strips and the same technology are used, gel matching is reproducibly possible. This is an important advancement for projects involving comparison of 2DE-gels produced over several years and in different laboratories.     

The transition from direct to delayed ionisation of C 60

The timescale for the coupling of electronic and vibrational excitation in isolated fullerenes is determined by recording positive ion time-of-flight mass spectra on excitation with ultrashort laser pulses at 790 nm of the same fluence but different pulse durations. The coupling leads to the onset of a delayed ionisation “tail” on the parent fullerene ion peak. This occurs for a pulse duration of 500-1000 fs, depending on laser fluence. Received 20 October 2000     

A unified expression of elastic–plastic notch stress–strain calculation in bodies subjected to multiaxial cyclic loading

In this paper, a simplified thermodynamics analysis of cyclic plastic deformation is performed in order to establish an energy transition relation for describing the elastic–plastic stress and strain behavior of the notch-tip material element in bodies subjected to multiaxial cyclic loads. Based on the thermodynamics analysis, it is deduced that in the case of elastic–plastic deformation, Neuber’s rule inevitably overestimates the actual stress and strain at the notch tip, while the equivalent strain energy density (ESED) method tends to underestimate the actual notch-tip stress and strain. According to the actual energy conversion occurring in the notch-tip material element during cyclic plastic deformation, a unified expression for estimating the elastic–plastic notch stress–strain responses in bodies subjected to multiaxial cyclic loads is developed, of which Neuber’s rule and the ESED method become two particular cases, i.e. upper and lower bound limits of the notch stress and strain estimations. This expression is verified experimentally under both proportional and non-proportional multiaxial cyclic loads and a good agreement between the calculated and the measured notch strains has been achieved. It is also shown that in the case of multiaxial cyclic loading, the unified expression distinctly improves the accuracy of the notch-tip stress–strain estimations in comparison with Neuber’s rule and the ESED method. The unified expression of the notch stress–strain calculation developed in this paper can thus provide a more logical approximate approach for estimating the elastic–plastic notch-tip stress and strain responses of components subjected to lengthy multiaxial cyclic loading histories for local strain approach-based fatigue-crack-initiation life prediction.     

Correlation between thermophoretic behavior and hydrophilicity for various alcohols⋆

The European Physical Journal E - In order to study the effect of cell elastic properties on the behavior of assemblies of motile cells, this paper describes an alternative to the cell phase field...     

PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF Euglena grcrcilis

Abstract —Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity Chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K_D= 4 n M ) as well as under reducing conditions in the presence of dithionite (K_d= 6 n M ). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately lo⁶ flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.     

Excited-state relaxation of protonated adenine.

The excited state dynamics of protonated adenine in the gas phase were investigated by femtosecond pump-probe transient mass spectroscopy. Adenine was protonated in an electrospray ionization source and transferred to a Paul trap. Two femtosecond laser pulses at 266 nm and 800 nm excited the lowest electronic pipi* state and probed the excited-state dynamics by monitoring ion fragment formation. The measured excited state decay is monoexponential with a lifetime shorter than 161 fs. This agrees with a theoretical prediction of very fast internal conversion via a conical intersection with the ground state.     

Femtosecond dynamics of electronic excitations of adsorbates studied by two-photon photoemission pulse correlation: CO/Cu(111)

An equal pulse correlation technique based on angle-resolved two-photon photoemission is employed to investigate the lifetime of electronic excitations of an adsorbate on a single crystal metal surface. Photoemission from an occupied surface state on Cu(111) by a non-resonant two-photon process via the sp-band gap is used to characterize directly at the surface the width and shape of 65 fs laser pulses at a photon energy of hv = 3.4 eV. The 2PPE correlation technique allows the establishment of an upper limit of τ < 20 fs for the lifetime of the 2π^* resonance of CO adsorbed on Cu(111), whereas from the spectral width of the 2π^* level a lower limit of ≈1 fs is estimated.     

Resonant two-photon ionisation spectroscopy of C 60

The electronic absorption spectrum of C ₆₀ has been measured using a continuous gas phase source which is capable of cooling the molecules to a vibrational temperature below ca. 100 K. The results are in good agreement with a previous high resolution gas phase spectrum reported for the spectral range 595 nm–630 nm, obtained with a pulsed Smalley source [1]. The spectral range down to 450 nm is reported for the first time for a molecular beam expansion and is compared with published spectra obtained in solution [2].