反应详情

To 25 ml aliquots of incubation buffer (0.01 M phosphate, 0.0027 M potassium chloride, 0.137 M sodium chloride, pH 7.4, Sigma Aldrich Corp, Catalog # P-4417), increasing amounts of solid phase cystine (Sigma Aldrich Corp, St Louis, Mo., Catalog # C-6195) were added and incubated for 48 hours at 37° C. with stirring. Residual solid phase was then separated to form a supernatant and a pellet, and the pellet was dissolved in 25 ml of a cystine-dissolving agent, an alkalinizing buffer of 0.1 M sodium carbonate, pH 9.9, (Sigma Aldrich Corp, Catalog # S-7795), to form a final cystine solution. Cystine concentration was determined in both the supernatant and the final cystine solution according to the procedure given in Example 3. Various separation techniques were used for removing the pellet: (1) in one experiment, centrifugation was performed at 4000 rpm at 37° C. for 20 minutes; (2) in two experiments, separation was performed by filtration using a Metricel filter (Gelman Sciences, Ann Arbor, Mich.); and (3) in four experiments, centrifugation was performed at room temperature at 3800 rpm for 20 minutes. As all methods were equivalent in outcome, the latter was employed in all subsequent experiments.

1. additionwere added
2. customResidual solid phase was then separated
3. customto form a supernatant
4. dissolutiondissolving agent