반응 #486546

반응 방정식

fluorescein isothiocyanate

N-hydroxysuccinimido biotin

→

fluorescein

Biotin

용매

phosphate buffered saline

dimethylformamide

dimethylsulfoxide

반응 조건

후처리

1. 1
   기타to react at room temperature for 4 hours
2. 2
   기타after which the unreacted reagents were quenched with 10 μl ethanolamine
3. 3
   기타The quenched reaction mixture
4. 4
   기타was then dialyzed
5. 5
   workup.DISTILLATIONagainst double distilled water until unreacted fluorescein
6. 6
   기타derivatives no longer dialyzed into the water

실험 절차

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.

출처