Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.     

A catalytic immuno-reactor for the amperometric determination of human serum albumin

A catalytic immuno-reactor for the determination of human serum albumin (HSA) was constructed by using immobilized antibody and an amperometric detector. A sandwich assay with hemin-labeled antibody to catalyze the decomposition of hydrogen peroxide was used, the catalytic activity of the hemin-antibody conjugate being determined by measuring the decrease in hydrogen peroxide concentration. The reaction of hemin-labeled antibody with antigen was complete within 30 min and the current decrease was correlated with the HSA concentration. The relative standard deviation was about 9% at an HSA concentration of 1 mg ml^?1.     

Flow injection analysis of potassium using an all-solid-state potassium-selective electrode as a detector

The concentration of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this laboratory demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h⁻¹). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.     

Rapid oligosaccharide synthesis using a fluorous protective group

The Bfp-OH, a novel fluorous protecting reagent, was able to be easily prepared. The Bfp group was readily introduced to a carbohydrate, removed in high yield, and recyclable after cleavage. The use of the Bfp group made it possible to synthesize a pentasaccharide by minimal column chromatography purification. Each synthetic intermediate was able to be easily purified only by simple fluorous-organic solvent extraction and monitored by TLC, NMR, and MS.     

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.