反応 #486546

反応方程式

fluorescein isothiocyanate

N-hydroxysuccinimido biotin

→

fluorescein

Biotin

溶媒

phosphate buffered saline

dimethylformamide

dimethylsulfoxide

反応条件

後処理

1. 1
   その他to react at room temperature for 4 hours
2. 2
   その他after which the unreacted reagents were quenched with 10 μl ethanolamine
3. 3
   その他The quenched reaction mixture
4. 4
   その他was then dialyzed
5. 5
   workup.DISTILLATIONagainst double distilled water until unreacted fluorescein
6. 6
   その他derivatives no longer dialyzed into the water

実験手順

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.

出典