Accurate detection of both glycoproteins and total proteins on blots: control of side reactions occurring after periodate oxidation of proteins.
The detection of glycoproteins based on the periodate oxidation of their carbohydrate moiety, and their conjugation to digoxigenin hydrazide directly on blots, leads to high background staining, especially with polyvinylidene difluoride (PVDF) membranes. The addition of Tween-20 to all incubation solutions, except at the oxidation step, strongly reduced the background staining and allowed us to detect lower amounts of glycans fixed on the blots through the protein moiety. The presence of polysaccharides during the oxidation step was shown to produce side reactions that led to the staining of nonglycosylated proteins; it demonstrated risks that may occur with crude extracts when periodate oxidation is performed in solution. The phenomenon was used to design a total protein-staining assay that can be included along with positive and negative controls in a general strategy based on blotting, which was delineated for the identification of glycoproteins in complex tissue extracts.
Bouchez-Mahiout
I
I
Laboratoire de Chimie Biologique, Centre INRA de Versailles-Grignon, Thiverval-Grignon, France.
Doyen
C
C
Laurière
M
M
D004076:Digoxigenin / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006023:Glycoproteins / Q000032:analysis*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting / Q000379:methods*; D008956:Models, Chemical; D010504:Periodic Acid / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D014908:Triticum / Q000737:chemistry
D006023:Glycoproteins; D011134:Polysaccharides; D011506:Proteins; D010504:Periodic Acid; D004076:Digoxigenin