Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.
Anal Biochem
; 372(1): 62-71, 2008 Jan 01.
Article
em En
| MEDLINE
| ID: mdl-17826731
ABSTRACT
Fluorescein isothiocyanate (FITC) is a well-known probe for labeling biologically relevant proteins. However, the impact of the labeling procedure on protein structure and biological activities remains unclear. In this work, FITC-labeled human plasma fibronectin (Fn) was developed to gain insight into the dynamic relationship between cells and Fn. The similarities and differences concerning the structure and function between Fn-FITC and standard Fn were evaluated using biochemical as well as cellular approaches. By varying the FITC/Fn ratio, we demonstrated that overlabeling (>10 FITC molecules/Fn molecule) induces probe fluorescence quenching, protein aggregation, and cell growth modifications. A correct balance between reliable fluorescence for detection and no significant modifications to structure and biological function compared with standard Fn was obtained with a final ratio of 3 FITC molecules per Fn molecule (Fn-FITC3). Fn-FITC3, similar to standard Fn, is correctly recruited into the cell matrix network. Also, Fn-FITC3 is proposed to be a powerful molecular tool to investigate Fn organization and cellular behavior concomitantly.
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Base de dados:
MEDLINE
Assunto principal:
Fibronectinas
/
Fluoresceína-5-Isotiocianato
/
Matriz Extracelular
Limite:
Animals
/
Humans
Idioma:
En
Ano de publicação:
2008
Tipo de documento:
Article