反应详情

A solution of 173 mg of keyhole limpet hemocyanin in 7 ml of 50 mM potassium phosphate (pH 7.5) was cooled in an ice-bath. To the solution were added 10.5 mL of dimethylsulfoxide (DMSO) dropwise, and the reaction temperature was maintained below room temperature. To the protein solution was added a solution of 40.2 mg of 1L in 1.5 mL of DMF dropwise. The mixture was allowed to stir at room temperature 18 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and dialyzed in 1 L of 70% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 50% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 30% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours) at room temperature. The trifluoroacetamido group of the conjugate was deprotected by dialysis of the resulting conjugate against 10% ammonium hydroxide for 3 days (1 L each for approximately 24 hours each), followed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C. (1 L each for at least 6 hours each). The protein concentration was determined to be 2.9 mg/mL using BioRad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248, 1976). A total of 34 mL of the conjugate was obtained. The extent of available lysine modification was determined to be 72% by the 2,4,6-trinitrobenzene sulfonate (TNBS) method (Habeeb AFSA, Anal. Biochem. 14:328-34, 1988).

1. temperaturethe reaction temperature was maintained below room temperature
2. customThe resulting conjugate was placed in a dialysis tube
3. custom(10,000 MW cut-off) and dialyzed in 1 L of 70% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 50% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 30% DMSO in 50 mM potassium phosphate (at least 3 hours)
4. custom1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours)
5. customat room temperature
6. customThe trifluoroacetamido group of the conjugate was deprotected by dialysis of the resulting conjugate against 10% ammonium hydroxide for 3 days (1 L each for approximately 24 hours each),
7. customfollowed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C.
8. customfor at least 6 hours
9. concentrationThe protein concentration
10. customA total of 34 mL of the conjugate was obtained