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.     

Formal catalytic asymmetric total synthesis of fostriecin

The common synthetic intermediate of a potent and promising anticancer agent, fostriecin, was synthesized using a unique method that combines four catalytic asymmetric reactions as shown above.     

Investigation of the triplet states of DNA by optical detection of magnetic resonance using a new slow passage technique

A new method to obtain slow passage ODMR signals under otherwise impeding conditions is described. The technique is applied to identify the triplet state of DNA as thymine. The zero field splitting constants measured are |D| = (0.203 ± 0.003) cm^?1, and |E| ? (0.0123 ± 0.0005) cm^?1. From microwave-induced delayed phosphorescence and slow passage measurements a preliminary kinetic scheme for the triplet state is derived.     

Dioxopyrrolines. LXII. Diels-Alder reaction of 1-Aryl-4- and 5-methoxycarbonyl-1H-pyrrole-2,3-diones with various 1,3-dienes

Two new dioxopyrrolines (1-aryl-4-methoxycarbonyl-1H-pyrrole-2,3-dione 6 and the 5-methoxycarbonyl isomer 8) behaved as good dienophiles to some kind of 1,3-dienes examined. In most cases, the products were explained by the reaction where the largest lobe of HOMO of dienes reacted to the larger LUMO of dienophiles in an expected cis-endo manner. However, in the reactions of 8 with alkylbutadienes, piperylene and isoprene, abnormality in the reaction was observed, which was well explained by taking account of steric factors.     

Synthesis of195mPt radiolabelled cis-diamminedichloroplatinum/II/ of high chemical and radiochemical purity using high performance liquid chromatography

An improved method is described for the synthesis of^195mPt-radiolabelled cis-diamminedichloroplatinum/II. An amount of 10 mg of 95% enriched¹⁹⁴Pt was irradiated for 75 h in the hydraulic conveyer of the KUR at a thermal neutron flux of approximately 8.15×10¹³ n.cm^–2.sec^–1 and the^195mPt-radiolabelled CDDP was purified using HPLC. The chemical yield is 61% its chemical purity is greater than 99.74% the radiochemical purity is nearly 100%, and the specific activity is 7.4×10⁶ Bq mg^–1 CDDP/200 Ci mg^–1 CDDP/.