fulltext.study @t Gmail

Covalent immobilization of epidermal growth factor molecules for single-molecule imaging analysis of intracellular signaling

Paper ID Volume ID Publish Year Pages File Format Full-Text
10573 692 2006 8 PDF Available
Title
Covalent immobilization of epidermal growth factor molecules for single-molecule imaging analysis of intracellular signaling
Abstract

We have developed cell stimulative system by covalently immobilized signalling molecules on the surface of coverslips on which cells are later cultured. N-(6-maleimidocaproyloxy)sulfo-succinimide (sulfo-EMCS) cross-links the amino-terminal of epidermal growth factors (EGF) with the thiol-modified glass surface without degrading EGF's physiological activity. The glass surface was covered up to about 1.0 EGF molecules nm−2 with uniform density. This density can be controlled by changing concentration of the maleimide-modified EGF in the solution reacting with the thiol-modified glass coverslips. When the density of EGF was only slightly lower than that of EGF receptor dimers, cellular response was dramatically decreased. The EGF receptor molecules bound with the immobilized EGF were prevented from lateral diffusion and internalization and kept their initial position. These properties are useful for quantitative, spatial and temporal control of the input signal stimulating cells in cellular signaling system studies. In addition, the immobility of the EGF in this system makes suitable targets for stable single-molecule observation under total internal reflection fluorescence microscopy (TIR-FM) to study EGF signalling mechanism, preventing lateral diffusion and internalization of EGF receptors. Here we show results of single-molecule observations of the association and dissociation between phosphorylated EGF receptors and Cy3-labeled growth factor receptor-binding protein 2 (Grb2) proteins in A431 cells stimulated by the immobilized EGF and discuss the utility of this method for the study of intracellular signal transduction.

Keywords
Cell activation; Cell signalling; Growth factors; Molecular imaging; Surface treatment
First Page Preview
Covalent immobilization of epidermal growth factor molecules for single-molecule imaging analysis of intracellular signaling
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us
Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 27, Issue 18, June 2006, Pages 3343–3350
Authors
, , , ,
Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us