Optimization of in-gel protein digestion system in combination with thin-gel separation and negative staining in 96-well plate format

Improvement of in-gel digestion efficiency is highly desirable for one- or two-dimensional gel electrophoretic separation and mass spectrometric (MS) analysis in proteomics, because the resultant increases in sequence coverage and MS signal intensity lead to higher confidence in protein identification. Here an optimized in-gel digestion system, in combination with thin-gel separation and negative staining in a high-throughput format using 96-well plates, is described. The combination of negative staining and protein separation on a 0.9 mm thick gel showed a clear improvement in in-gel digestion efficiency in comparison with the more typical protocols such as the combination of silver staining and a 1.0 mm gel. In addition, the use of 96-well plates to increase throughput did not decrease the efficiency of this strategy when the stirring of the gel pieces in processes such as destaining, washing, gel-shrinking and peptide extraction was performed by sonication instead of shaking the plates. This procedure was optimized and applied to identify proteins of the postsynaptic density fraction; 105 proteins were identified after SDS-PAGE separation.     

Synthesis of polyamides by the polyaddition of bisimidazoline with dicarboxylic acids

N,N′-Dipropionylethylenediamine was synthesized by the ring-opening addition reaction of 2-ethyl-2-imidazoline with propionic acid at 220°C. By applying this reaction to polymerization, polyamides were synthesized by the ring-opening polyaddition reaction at 220°C. of 1,4-bis(imidazoline-2-yl)butane with adipic acid, succinic acid, sebacic acid, and terephthalic acid. The reaction product of 1,4-bis(imidazoline-2-yl)butane with adipic acid, which was proposed to be nylon 26, was compared with an authentic sample of nylon 26 and shown to possess a very similar infrared spectrum and melting point.     

Conversion of endohedral <Superscript>133</Superscript>Xe-fullerene to endohedral <Superscript>133</Superscript>Xe-fullerenol to be used in nuclear medicine

Summary Hydrophilic endohedral ¹³³Xe-fullerenols, [¹³³Xe@C₆₀(OH)_xand ¹³³Xe@C₇₀(OH)_x], were synthesized from hydrophobic endohedral ¹³³Xe-fullerenes. The yield of endohedral ¹³³Xe-fullerenols extracted in water was about 40% and 23% for C₆₀and C₇₀, respectively. The products stored in 0.9% NaCl solution at 20 °C were stable enough to be used in nuclear medicine.     

Reaction of carbon monoxide with N-lithioketimines derived by the addition of alkyllithium to ortho-substituted benzonitriles. Diverse courses of cyclization via carbonyl anions

N-Lithioketimines, generated in situ from 2-substituted benzonitriles and t-BuLi, reacted with carbon monoxide to afford five-, six- or seven-membered cyclic products, involving participation by the ortho substituents. The reactions represent new selective transformations of carbonyllithium intermediate which previously were observed to lead to complicated reactions.     

Determination of the change of nuclear charge radius of the119Sn Mössbauer transition by internal conversion

Radioactive¹¹⁹Sb was implanted into six host matrices (CaSnO₃, Pt, Y, Au,-Sn, Pb) and internal conversion electrons of the 23.87 keV transition in¹¹⁹Sn were measured with an iron-free magnetic spectrometer as well as Mössbauer spectra. In the analysis of the conversion spectra of outermost electrons, the overlapping K-LM Auger lines were subtracted using the Auger spectrum of tin measured with another source of^117mSn, and the shake-off effect accompanying the conversion process was considered. From the correlation between the Mössbauer isomer shifts and the intensity ratios of O-shell to N₁-shell conversion electrons, the change of the nuclear charge radius of the 23.87 keV transition of¹¹⁹Sn was deduced to be R/R=(0.87 ± 0.25) × 10^–4 for a uniform charge distribution ofR= 1.2 ×A ^1/3 fm or, equivalently, r²>—=(3.6 ± 1.0) ×10^–3 fm².     

Nuclear-related analytical techniques: Literature retrieval from the INIS database

The applicability of the INIS database for literature retrieval on nuclear-related analytical techniques [e.g. neutron activation analysis (NAA), X-ray fluorescence (XRF), particle-induced X-ray emission (PIXE)] has been studied. There exist about 9700 documents on NAA and other forms of activation analysis, about 3500 documents on XRF and about 1200 documents on PIXE in the database collected from 1976 until July 1988. They cover a variety of elements and matrices in the fields of biology, geochemistry and the physical sciences. Some technical notes and comments on the practical use of this database are also included.     

Preparation of immunoaffinity mini-columns for the analysis of platelet activating factor (PAF) in biological samples.

Using an antibody to BN 52719, an analogue of platelet activating factor (PAF), immunoaffinity mini-columns for the separation of PAF from biological samples were prepared. Rabbits were immunized with BN 52719 and immunoglobulin G (IgG) from the antiserum was coupled with Sepharose 4B. The resulting suspension of the IgG-coated Sepharose 4B in 25 mM phosphate buffer (pH 6.9) was poured into a plastic mini-column (bed volume 2.0 x 0.8 cm). Stepwise elution of the column with methanol revealed that lyso-PAF is eluted with 20-30% methanol in water whereas PAF is eluted with 50-80% methanol. For the determination of PAF in biological samples, it is recommended that lipids are extracted from the samples and the extract, reconstituted in 20% methanol, is loaded on the column. The column is then washed with 50% methanol followed by elution of PAF with 80% methanol. A small amount of [3H]PAF is added to the samples for measurement of the recoveries of PAF during the procedures of extraction and elution. The PAF is then quantified by radioimmunoassay or bioassay. Employing the immunoaffinity mini-column and radioimmunoassay, the contents of PAF in macrophages and conditioned medium after stimulation with calcium ionophore A23187, or tumor promoters such as TPA and thapsigargin, were measured.     

High n-value phases in the complex bismuth oxides with layered structure,Bi2CaNan−2NbnO3n+3

Complex bismuth oxides with layered structure are prepared with a series of compositions in the system Bi₂CaNb₂O₉-NaNbO₃. It is found by X-ray powder diffraction that each compound is composed of more than two phases, which are described by a formula Bi₂CaNa_n?2Nb_nO_3n+3, e.g., in the sample with the nominal composition Bi₂CaNb₂O₉ · 8NaNbO₃, the phases with n = 6 to 8 appear predominantly. These phases are closely intergrown to each other. Moreover, high-resolution electron microscopy reveals that microsyntactic intergrowth frequently occurs in the phases with n > 5. The occurrence of the latter intergrowth is explained in terms of the bond length obtained.     

Production of endohedral 133Xe-fullerene by ion implantation

Ion implantation was applied to the production of endohedral ¹³³Xe-fullerene. Using an isotope separator, ¹³³Xe ions were implanted into a fullerene target of C₆₀ and C₇₀ produced by vacuum evaporation on a Ni backing. An HPLC analysis following dissolution of the fullerene targets in o-dichlorobenzene corroborated the formation of ¹³³Xe@C₆₀ and ¹³³Xe@C₇₀, showing a strong correlation between C₆₀/C₇₀ and ¹³³Xe. The observed tailing following ¹³³Xe peaks in the elution curves suggests a possibility of the isolation of endohedral ¹³³Xe-fullerene from empty fullerene.