反应详情

A solution of 188 mg of keyhole limpet hemocyanin (KLH) in 5.5 ml of 50 mM potassium phosphate (pH 7.5) was cooled in an ice-bath. To the solution were added 6 mL of dimethylsulfoxide dropwise, and the reaction temperature was maintained below room temperature. Then a solution of 54 mg (0.10 mmol) of 2L in 1.2 mL of DMF was added to the protein solution 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 was 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 2.1 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 60% by the 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 was 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