Improved binding of acidic bone matrix proteins to cationized filters during solid phase assays.
A number of commercially available matrix filter supports have been designed for the immobilization of proteins following either electrotransfer from sodium dodecyl sulfate (SDS) polyacrylamide gels or direct application during dot blotting assays. These matrices differ with respect to chemical composition, charge, pore size, and degree of hydrophobicity. It follows that the properties of the protein(s) of interest will greatly influence the degree to which they interact with and ultimately bind to various filters. Acidic bone proteins contain diverse post-translational modifications that influence their interactions with solid phase matrices such as those used in immunoblotting (Western or dot blotting) or ion binding (overlay) procedures. This communication describes the results of a study comparing binding of various mixtures of non-collagenous acidic bone matrix phosphoproteins as well as purified osteopontin and osteocalcin to various filters including nitrocellulose and cationized paper or nylon. Based on our findings, we recommend the use of cationized filters for solid phase assays requiring the binding of these acidic macromolecules to background supports.
Farach-Carson
M C
MC
Department of Biological Chemistry, University of Texas Dental Branch, Houston 77225.
Wright
G C
GC
Butler
W T
WT
D000818:Animals; D001857:Bone Matrix / Q000737:chemistry*; D002412:Cations; D003094:Collagen / Q000032:analysis / Q000737:chemistry; D003101:Collodion; D005374:Filtration / Q000295:instrumentation*; D006023:Glycoproteins / Q000032:analysis* / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D009757:Nylons; D015676:Osteonectin / Q000032:analysis / Q000737:chemistry; D053495:Osteopontin; D010209:Paper; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D012795:Sialoglycoproteins / Q000032:analysis / Q000737:chemistry
D002412:Cations; D006023:Glycoproteins; D009757:Nylons; D015676:Osteonectin; D010750:Phosphoproteins; D012795:Sialoglycoproteins; C506897:Spp1 protein, rat; D053495:Osteopontin; D003101:Collodion; D003094:Collagen