Reaction #64837

Reaction equation

fluorescein isothiocyanate

N-hydroxysuccinimido biotin

→

Biotin

fluorescein

Solvents

phosphate buffered saline

dimethylformamide

dimethylsulfoxide

Conditions

Workup

1. 1
   Otherto react at room temperature for 4 hours
2. 2
   Otherafter which the unreacted reagents were quenched with 10 μl ethanolamine
3. 3
   OtherThe quenched reaction mixture
4. 4
   Otherwas then dialyzed
5. 5
   workup.DISTILLATIONagainst double distilled water until unreacted fluorescein
6. 6
   Otherderivatives no longer dialyzed into the water

Procedure

Biotin and fluorescein labeled insulin was prepared. To 1 ml of a 1 mg/ml solution of insulin protein in phosphate buffered saline was added simultaneously 100 μl of a 1 mg/ml solution of fluorescein isothiocyanate (FITC) in dimethylformamide (DMF) and 100 μl of a 1 mg/ml solution of N-hydroxysuccinimido biotin in dimethylsulfoxide (DMSO). The two labeling reagents were allowed to react at room temperature for 4 hours, after which the unreacted reagents were quenched with 10 μl ethanolamine. The quenched reaction mixture was then dialyzed against double distilled water until unreacted fluorescein derivatives no longer dialyzed into the water. The covalent attachment of biotin and fluorescein to the desired protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis.

Source