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• W2077981036 abstract "A method is described for determination of carbohydrate and protein contents of glycoproteins separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electroblotted onto polyvinylidene difluoride (PVDF) membranes. Blots were stained, and appropriate pieces of PVDF membranes were excised, destained, and subjected to sequential hydrolysis with 0.2 M trifluoroacetic acid (TFA) for 1 h at 80°C, then with 2 M TFA for 4 h at 100°C, and finally with 6 M HCl at 100°C for 24 h to release sialic acids, neutral sugars with hexosamines, and amino acids, respectively. In some instances preliminary methanolysis was used. Carbohydrates including sialic acids were quantitated by high pH anion exchange chromatography with pulsed amperometric detection. Protein content of the bands was determined as amino acids by the fluorescamine or ninhydrin method. In the calculation of results proper adjustments were made for small amounts of fucose released by hydrolysis with 0.2 M TFA at 80°C, and for partial degradation of protein during hydrolysis with 2 M TFA at 100°C. Recoveries of amino acids from hydrolysates of glycoproteins that had been electroblotted onto PVDF membranes equaled those of carbohydrates. This was possible because of preliminary hydrolysis of glycoproteins with TFA, as well as washing of wet, instead of dried, PVDF membranes after hydrolysis with 6 M HCl. The two modifications increased yields of amino acids by about 30%. The method was successfully applied to the determination of molar and weight percentage composition of human transferrin, band 3 protein, glycophorin A, and α1-acid glycoprotein. In each case the results obtained for directly hydrolyzed and electrophoresed/electroblotted glycoproteins were practically identical. We also determined the glucosamine content of band 4.1 protein of erythrocytes." @default.
• W2077981036 created "2016-06-24" @default.
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• W2077981036 date "1999-11-01" @default.
• W2077981036 modified "2023-10-16" @default.
• W2077981036 title "A Single-Sample Method for Determination of Carbohydrate and Protein Contents Glycoprotein Bands Separated by Sodium Dodecyl Sulfate– Polyacrylamide Gel Electrophoresis" @default.
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• W2077981036 doi "https://doi.org/10.1006/abio.1999.4294" @default.
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