Title
Characterization of the osteogenic stromal cell line <tex>$MN_{7}$</tex> : identification of secreted <tex>$MN_{7}$</tex> proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing Characterization of the osteogenic stromal cell line <tex>$MN_{7}$</tex> : identification of secreted <tex>$MN_{7}$</tex> proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences . Biomedical Sciences
Publication type
article
Publication
New York, N.Y. ,
Subject
Human medicine
Source (journal)
Journal of bone and mineral research. - New York, N.Y.
Volume/pages
9(1994) :6 , p. 903-913
ISSN
0884-0431
ISI
A1994NN31400015
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the a, chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.
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