| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
We have demonstrated the separation of human serum proteins with the Beckman P/ACE 2100 Capillary electrophoresis (CE) system (Beckman Inc., Brea, CA,USA). A 20 cm uncoated fused silica capillary column (25mum, I.D.) and 150 mmol/L borate buffer (pH 10.0) as the running buffer were used. Human serum samples were diluted 11-folds in 20 mmol/L PBS buffer (pH 7.0) before application to CE column by pressure injection for 10 second (approximately 10 nL). The CE column temperature was maintained at 23ﾟC.A voltage of 10kV was applied for 6.5 min separating the protein fractions and the peak were detected at 200 nm. Human serum sample was fractionated into approximately 10 fractions by the proposed method. We confirmed the validity of our identifying the proteins fractioned, employing method for separating serum proteins by affinity column and antibody identification procedure. These protein peaks were identified a immunoglobulin, complement C3, transferin, alpha 2-macroglobulin, haptoglobin,
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alpha 1-antitrypsin, albumin, prealbumin, respectively, according to increasing migration time with CE.Moreover, the results of analyzes of 38 samples using the proposed method correlated well with those obtained by the cellulose acetate electrophoresis method (Olympus Optical Co.Ltd, Tokyo, Japan). We also have developed separation and quantitative estimation of the isoenzymes of lactate dehydrogenase in human serum were accomplished with Beckman P/ACE 2100 capillary electrophoresis method. A uncoated fused silica column 50 cm long, 75 mum I.D.and substrate containing running buffer [51.6 mmol/L L-lactatic acid, 8.26 mmol/L NAD in 25 mmol/L Tris (hydroxymethyl) aminoethanol buffer, pH 8.7] were used. The resulting product NADH was detected at 340 nm. Injecting a sample (serum samples diluted 5 times by 25 mmol/L Tris buffer (pH8.7) by pressure injection for 2 seconds. Separating the isoenzymes with 10kV applied for 5 min, turning off the voltage for 30 min of incubation at 24ﾟC (for reaction between substrate and isoenzymes), and reapplying 10 kV 30 min. Under these conditions, the NADH generated by LD5 emerged at 20 min, the LD-1 peak 23.5 min with closed to baseline separation of the other isoenzymes which emerge between LD-5 to LD-1 peak, another peak appears, termed Sample Shock The results obtained by the proposed CE method correlated well those by REP (Helena Labs, T.X., USA) gel electrophoresis. In conclusion, proposed proteins analysis and lactate dehydrogenase isoenzymes analysis by CE system is sensitive, precise, easy for clinical use. Less
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