Combined confocal and magnetic resonance microscopy

Confocal fluorescence optical microscopy and magnetic resonance microscopy are each used to study live cells in a minimally invasive way. Both techniques provide complementary information. Therefore, by examining cells simultaneously with both methodologies, more detailed information is obtained than is possible with each microscope individually. In this paper two configurations of a combined confocal and magnetic resonance microscope are described. The first configuration is capable of studying large single cells or three-dimensional cell agglomerates, whereas the second configuration is designed for the investigation of monolayers of mammalian cells. In both cases the sample compartment is part of a temperature regulated perfusion system. Images obtained with the combined system are shown forXenopus laevis oocytes, model JB6 tumor spheroids, and a single layer of Chinese hamster ovary cells. Finally, potential applications of the combined microscope are discussed.     

Identification of phage antibodies toward the Werner protein by selection on Western blots

A procedure was established for selecting phage antibodies (phage-abs) from phage-displayed antibody repertoires by panning against proteins, separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto nitrocellulose membranes (Western blots). This immobilization strategy is applicable for secondary rounds of panning in selections against semipurified proteins, and directs the selection toward antibodies suitable as immunochemical reagents in Western blots. In model experiments, enrichment factors as high as 1.9x10(5) were obtained in a single round of panning. Furthermore, we demonstrate the application of this approach by selection of phage-abs recognizing the human Werner protein, which is defective in a premature aging syndrome.     

Proton affinity of β-oxalylaminoalanine (BOAA): Incorporation of direct entropy correction into the single-reference kinetic method

A new version of the single-reference-extended kinetic method is presented in which direct entropy correction is incorporated. Results of calibration experiments with the monodentate base pyridine and the bidentate base ethylenediamine are presented for which the method provides proton affinities in excellent agreement with published values and reasonable predictions for the protonation entropies. The method is then used to determine the proton affinity and protonation entropy of the non-protein amino acid beta-oxalylaminoalanine (BOAA). The PA of BOAA is found to be 933.1 +/- 7.8 kJ/mol and a prediction for the protonation entropy of -39 J mol(-1) K(-1) is also obtained, indicating a significant degree of intramolecular hydrogen bonding in the protonated form. These results are supported by hybrid density functional theory calculations at the B3LYP/6-311++G**//B3LYP/6-31+G* level. They indicate that the preferred site of protonation is the alpha-nitrogen atom (PA = 935.0 kJ/mol) and that protonated BOAA has a strong hydrogen bond between the hydrogen on the alpha-amino group and one of the carbonyl oxygen atoms on the side chain.     

EPR and 1H and 13C dynamic nuclear polarization studies of a molecularly doped polymer: bisphenol A polycarbonate doped with trianisylamine and trianisylaminium perchlorate.

We have investigated the EPR and DNP behavior of a molecularly doped polymer modeling those used to transport electronic charge in electrophotography. The EPR spectra show no evidence of the superexchange reported for a closely related system based on tri-p-tolylamine. The difference may be due to larger charge-transfer matrix elements in the latter system. An unambiguous interpretation of the observed 1H DNP was rendered difficult by the unanticipated asymmetry of the EPR spectra. We report extensive data on the "three-spin" effect evident in the DNP-enhanced 13C NMR spectra, and comment on its potential for characterizing polymer interfaces.     

13C solid state NMR investigation of structural relaxation of the polymer backbone in poly (ethylmethacrylate)

Geometry and time scale of structural relaxation of poly(n-alkylmethacrylates) above the glass transition is studied by temperature dependent one- and two-dimensional 13C-NMR spectroscopy. The geometry of the isotropization of the polymer backbone as deduced from detailed analysis of spectral line shapes is identified as random angular jumps. Analysis of echo decays confirms that at a given temperature this isotropization can adequately be described with a single correlation time. The results are discussed in terms of conformational memory and local structure recently identified in these polymeric glasses.     

Investigation into factors affecting precision in ion trap mass spectrometry using different scan directions and axial modulation potential amplitudes

Electrospray ionization mass spectra obtained from different scan directions are observed to be dependent on the axial modulation potential amplitudes used for resonant ejection and on the positive deviation caused by higher even-multipole fields present in most commercial ion traps. The axial modulation voltage influences the dissociation of ions during resonant ejection and the observed mass shifts. The higher even-multipole fields in commercial ion traps are known to influence resonant ejection from the ion trap and can cause a loss in mass resolution for peaks in reverse scan mass spectra compared with that obtained by the forward scan. However, along with the dissociation of ions during resonant ejection causing a loss in resolution, the possibility of resolving an isotopic distribution is also shown to be influenced by the mass shifts caused by the space charge. These mass shifts differ depending on the scan direction employed. A significant loss in resolution can also result from resonant ejection using non-optimal axial modulation voltages. We also present results showing the ejection of ions at betaz = 1/2 using the reverse scan mode without the axial modulation voltage. Ion ejection at betaz = 1/2 is uncommon in commercial (stretched ion traps) with the conventional analytical scan without the use of a frequency of the axial modulation voltage corresponding to this non-linear resonance.     

Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation-element mass spectrometry with phosphorus-31 detection.

Laser ablation inductively coupled plasma-mass spectrometry (ICP-MS) with (31)P detection has been used for spotting of phosphoproteins after one-dimensional polyacrylamide gel electrophoresis (1-D PAGE) and membrane transfer. By analyzing a mixture of myoglobin, alpha-casein and reduced fibrinogen it is demonstrated that phosphoproteins are specifically recognized by this method. A special washing step was found to be necessary to remove phosphate noncovalently bound to proteins. The (31)P signal was found to contain quantitative information both with respect to relative and absolute amounts of phosphorus present in phosphoproteins. Normalizing the (31)P signal from a single laser ablation trace by the total amount of phosphoprotein applied to the gel, a detection limit of 5 pmol of phosphorus is estimated.