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The resulting solution was desalted using three PD-10 columns (Amersham Pharmacia Biotech) to produce 5.5 mL of pooled protein solution. This solution was cooled to 0° C., and 4 mL of DMSO was added dropwise. A solution of 7 mg (0.014 mmol) of MDEA-maleimido derivative (2O) in 0.5 mL of DMSO was added to the protein solution. The mixture was allowed to stir at room temperature for 24 hours. To the protein solution was added 400 μL of 5 mg/mL ethyl maleimide in DMSO to quench any unreacted thiol groups, and the mixture was allowed to stir at room temperature for 24 hours. The resulting conjugate was placed in a dialysis tube (10,000 MW cut-off) and was dialyzed in 1 L of 30% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 20% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours), 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 0.9 mg/mL using BioRad Coomassie blue protein assay (Bradford, M., Anal. Biochem. 72:248,1976). A total of 15 mL of the conjugate was obtained.

1. customto quench any unreacted thiol groups
2. stirringto stir at room temperature for 24 hours
3. customThe resulting conjugate was placed in a dialysis tube
4. custom(10,000 MW cut-off) and was dialyzed in 1 L of 30% DMSO in 50 mM potassium phosphate (pH 7.5, 3 changes, at least 3 hours each), 1 L of 20% DMSO in 50 mM potassium phosphate (at least 3 hours), 1 L of 10% DMSO in 50 mM potassium phosphate (at least 3 hours)
5. customfollowed by 6 changes with 50 mM potassium phosphate (pH 7.5) at 4° C.
6. customfor at least 6 hours
7. concentrationThe protein concentration
8. customA total of 15 mL of the conjugate was obtained