Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes.
A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.
Thornton
D J
DJ
Department of Biochemistry & Molecular Biology, University of Manchester, United Kingdom.
Holmes
D F
DF
Sheehan
J K
JK
Carlstedt
I
I
D003101:Collodion; D003124:Colorimetry; D004586:Electrophoresis / Q000379:methods; D008567:Membranes, Artificial; D008832:Microchemistry / Q000379:methods; D009077:Mucins / Q000032:analysis*; D010446:Peptide Fragments / Q000032:analysis; D010503:Periodic Acid-Schiff Reaction; D012015:Reference Standards
D008567:Membranes, Artificial; D009077:Mucins; D010446:Peptide Fragments; D003101:Collodion