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

HRID 36559

Reaktionsgleichung

Reaktionsgleichung von HRID 36559
[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

Komponenten

7

Edukt 2

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

Produkt 2

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

Reagenzien, Katalysatoren, Lösungsmittel 3

HCID 679Dimethyl SulfoxideLösungsmittelHCID 6228DimethylformamideLösungsmittelHCID 24978514Phosphate Buffered SalineLösungsmittel

Versuchsvorschrift

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.

Aufarbeitung

  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

Quellen und Nachweise

ORD-Eintrag
ord-58ea642946594e2098e864896dd7741c
Quelldatensatz
uspto-grants-1995_05
Patentnummer
US05416016

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