ANALYSIS OF EXTRACELLULAR MATRIX PROTEINS PRODUCED BY CULTURED CELLS
L. V. Turoverova,1,* M. G. Khotin,1 N. M. Yudintseva,1 K.-E. Magnusson,2 M. I. Blinova,1
G. P. Pinaev,1 D. G. Tentler 1,*
1 Institute of Cytology RAS, and 2 Linkoping University, Department of Clinical and Experimental Medicine, Sweden;
* e-mail: lvtur@mail.ru;
dtentler@mail.ru
Extracellular matrix (ECM) is a highly organized multimolecular structure essential for vital function of
any organism. Although a lot of data on the extracellular matrix components has been accumulated, an isolation
of the entire set of these proteins still remains to be a complex procedure since ECM contains fibrillar proteins
and proteoglycans, which form multidomain net-like structures. In the presented study, we developed a method
for isolation of ECM proteins from cell cultures. Human epidermoid carcinoma cells A431 and fibroblasts obtained
from normal and scar human skin were used. We showed that EDTA solution removed cells from culture
plates without destroying the cell membrane. Following treatment of remaining ECM proteins with acetic acid
in order to dissociate collagen fibrils significantly improved fractioning of ECM proteins. Extraction of the remained
proteins from culture plate surface was preformed using a buffer developed on the basis of Laemmli probe
buffer. With this method, we isolated ECM proteins synthesized by culturing cells and suitable for a future
analysis by SDS PAGE and two-dimentional electrophoresis as well as for identification of individual proteins
by mass-spectrometry. This study may allow comparing protein contents of ECMs isolated from different sources,
and elucidate influences of various proteins on the protein and the properties of extracellular matrix of investigated
cells.
Key words: extracellular matrix, fibroblasts, collagen, fibronectin
| Pdf |
Back |
Contents |
Main |
|
