Detection of glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis using the periodic acid-Schiff stain.
We observed that published methods for staining glycoproteins in sodium dodecyl sulfate-polyacrylamide gels did not stain glycoproteins separated by nondenaturing polyacrylamide gel electrophoresis. Therefore, the periodic acid-Schiff stain for glycoprotein was adapted for use with proteins analyzed by nondenaturing polyacrylamide gel electrophoresis. Following nondenaturing polyacrylamide gel electrophoresis proteins were denatured in situ by incubation with aqueous 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 40% ethanol, and 5% acetic acid at 90 degrees C for 15 min followed by periodic acid-Schiff staining. This modified procedure will detect at least 0.2 micrograms protein-associated carbohydrate. Omission of the periodic acid treatment from the protocol was used as a control to detect nonspecific staining of some proteins. This modified procedure was validated using both purified glycoproteins and extracellular culture fluid containing carbohydrate-associated proteins of Ruminococcus flavefaciens.
Doerner
K C
KC
Department of Animal Sciences, University of Illinois, Urbana 61801.
White
B A
BA
D001431:Bacteriological Techniques; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006023:Glycoproteins / Q000032:analysis*; D006651:Histocytochemistry; D010503:Periodic Acid-Schiff Reaction; D011489:Protein Denaturation; D012394:Rosaniline Dyes
D003470:Culture Media; D006023:Glycoproteins; D012394:Rosaniline Dyes; C004692:coomassie Brilliant Blue