The Conjugation of Fluorescein Isothiocyanate to Annexin V and its Application
- VernacularTitle:膜联蛋白V的异硫氰酸荧光素标记和应用研究
- Author:
Jiong CAI
;
Fang LI
;
Na NIU
;
Shizhen WANG
- Publication Type:Journal Article
- Keywords:
Annexin V;
FITC;
Pichia pastoris;
Phosphatidylserine
- From:
Journal of Medical Research
2006;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To prepare a novel annexin V and conjugate it with fluorescein isothiocyanate and test their binding ability to phosphatidylserine(PS)exposed erythrocytes.Methods The annexin V fused with an metal chelating binding site was obtained from pichia pastoris culturing and methanol induction expression.The annexin V was purified from the culture supernatant crude product by ion-exchange chromatography and ammonium sulfate precipitation.The annexin V was conjugated with fluorescein isothiocyanate in boric buffer and the conjugate annexin V-FITC was purified by ion-exchange chromatography.Sheep red blood cells were treated with Glutaraldehyde to expose membrane PS.The annexin V-FITC binding to PS exposed erythrocytes was investigated by fluorescent microscopy.Results The novel annexin V was purified and concentrated from pichia pastoris culture by ion-exchange chromatography and ammonium sulfate precipitation.After conjugating with FITC,the annexin V was found to bind PS exposed erythrocytes by fluorescent microscopy.Conclusion The annexin V expressed recombinantly in pichia pastoris retains the binding ability to PS exposed erythrocytes and is applicable to apoptosis detection in vitro.
