Fluorescein 5-isothiocyanate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

HRID 36559

반응식

HRID 36559 반응식
[CH:1]1[C:6](N=C=S)=[CH:5][C:4]2[C:10]([O:12][C:13]3([C:23]4[CH:24]=[CH:25][C:26]([OH:28])=[CH:27][C:22]=4[O:21][C:15]4[CH:16]=[C:17]([OH:20])[CH:18]=[CH:19][C:14]3=4)[C:3]=2[CH:2]=1)=[O:11].O[N:30]1[C:51](=[O:52])[NH:50][C@H:49]2[C@@H:31]1[CH2:32][S:33][C@H:34]2[CH2:35][CH2:36][CH2:37][CH:38](N1C(=O)CCC1=O)[C:39](=[O:41])[OH:40]>OP([O-])(O)=O.OP([O-])([O-])=O.[Na+].[Na+].[Na+].[Cl-].[Cl-].[K+].[K+].CN(C)C=O.CS(C)=O>[OH:41][C:39]([CH2:38][CH2:37][CH2:36][CH2:35][C@H:34]1[C@@H:49]2[C@@H:31]([NH:30][C:51]([NH:50]2)=[O:52])[CH2:32][S:33]1)=[O:40].[CH:1]1[CH:6]=[CH:5][C:4]([C:10]([OH:12])=[O:11])=[C:3]([C:13]2[C:14]3[CH:19]=[CH:18][C:17]([OH:20])=[CH:16][C:15]=3[O:21][C:22]3[C:23]=2[CH:24]=[CH:25][C:26]([CH:27]=3)=[O:28])[CH:2]=1

참가 물질

7

반응물 2

HCID 18730Fluorescein 5-isothiocyanateHCID 129676281N-Hydroxysuccinimido-d-biotin

생성물 2

HCID 33832-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acidHCID 171548Biotin

시약·촉매·용매 3

HCID 679Dimethyl Sulfoxide용매HCID 6228Dimethylformamide용매HCID 24978514Phosphate Buffered Saline용매

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.

후처리

  1. customto react at room temperature for 4 hours
  2. customafter which the unreacted reagents were quenched with 10 μl ethanolamine
  3. customThe quenched reaction mixture
  4. customwas then dialyzed
  5. distillationagainst double distilled water until unreacted fluorescein
  6. customderivatives no longer dialyzed into the water

출처 및 근거

ORD 기록
ord-58ea642946594e2098e864896dd7741c
출처 데이터셋
uspto-grants-1995_05
특허 번호
US05416016

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