反应详情

A solution of 500 mg of bovine serum albumin (BSA) in 6.7 mL of 50 mM potassium phosphate (pH 7.5) was cooled in an ice-bath. To the solution was added 8.5 mL of DMSO dropwise, and the reaction mixture was maintained below room temperature. To the protein solution was added a solution of 12 mg (0.022 mmol) of 2L in 1.5 mL of anhydrous DMF dropwise. The reaction mixture was allowed to stir at room temperature 48 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). The protein concentration was determined to be 7.12 mg/mL using BioRad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248,1976). A total of 45 mL of the conjugate was obtained.

1. temperaturethe reaction mixture 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. concentrationThe protein concentration
9. customA total of 45 mL of the conjugate was obtained