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Controlled coupling of mildly reduced proteins to Sepharose gelatin by heterobifunctional reagent |
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| Authors: | AFSA Habeeb |
| Institution: | Department of Biochemistry and Nutrition, Medical Sciences Campus, University of Puerto Rico, GPO Box 5067, San Juan, Puerto Rico 00936 USA |
| Abstract: | The heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate was utilized for controlled coupling of mildly reduced BSA and lysozyme to Sepharose gelatin to prepare immunoabsorbents. Each reaction step was examined and quantitated. The free amino groups on gelatin after coupling gelatin to cyanogen bromide-activated Sepharose as well as the number of 3-(2-pyridyldithio) propionyl groups on Sepharose gelatin were quantitated. Coupling of mildly reduced BSA as well as lysozyme to PDP-Sepharose gelatin occurred through sulfhydryl-disulfide exchange and permitted the formation of an immunoabsorbent. The immunoabsorbents were capable of binding the respective antibody and the eluted antibodies were pure and free of plasma proteins. |
| Keywords: | SPDP N-succinimidyl 3-(2-pyridyl dithio) propionate PDP 3-(2-pyridyl dithio) propionyl TP thiopropionyl Bis N N′-methylene bisacrylamide TEMED N N N′ N′-tetramethylethylenediamine DTT dithiothreitol TNBS trinitrobenzene sulfonic acid TNP trinitrophenyl PAGE polyacrylamide gel electrophoresis CNBr cyanogen bromide DTNB 5 5′-dithiobis (2-nitrobenzoic acid) SDS sodium dodecyl sulfate |
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