[Analysis of extracellular matrix proteins produced by cultured cells].
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.
L V
LV
Turoverova
M G
MG
Khotin
N M
NM
Iudintseva
K E
KE
Magnusson
M I
MI
Blinova
G P
GP
Pinaev
D G
DG
Tentler
D019342:Acetic Acid; D045744:Cell Line, Tumor; D004492:Edetic Acid; D015180:Electrophoresis, Gel, Two-Dimensional; D016326:Extracellular Matrix Proteins / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D012967:Sodium Dodecyl Sulfate; D014325:Tromethamine
D016326:Extracellular Matrix Proteins; C088816:Laemmli buffer; D014325:Tromethamine; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D019342:Acetic Acid