Quantification of fibronectin matrix assembly sites using a novel ELISA assay.
Binding of the N-terminus of fibronectin to assembly sites on the cell surface is an essential step in fibronectin fibrillogenesis. Fibronectin matrix assembly sites have customarily been quantified using an iodinated 70 kDa N-terminal fibronectin fragment. The 125I-70 K fragment is a less than ideal reagent because its preparation requires large amounts of plasma fibronectin and it has a fairly short shelf life. An additional limitation is that the cells responsible for binding the 125I-70 K cannot be quantified or identified directly but must be assessed in parallel cultures. To overcome these disadvantages, we developed an ELISA-based assay using a recombinant HA-tagged 70 K fragment. This assay allows for the simultaneous quantification and localization of matrix assembly sites on the surface of adherent cells.
Zheng
Mingzhe
M
Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland Avenue, Albany, New York 12208, USA.
Ambesi
Anthony
A
Yu
Lin
L
McKeown-Longo
Paula J
PJ
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D017434:Protein Structure, Tertiary; D019096:Vitronectin / Q000737:chemistry
D005353:Fibronectins; D019096:Vitronectin