Artifacts following gold staining of Western-blotted membranes.
Protein samples were separated by sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and stained with colloidal gold. Three artifact bands appeared demonstrating apparent molecular weights of 58,000, 63,000, and 65,000. The appearance of these bands was due to oxidized dithiothreitol used to denature the proteins prior to electrophoresis. The appearance of the artifact bands could be avoided by the addition of excess iodoacetamide to the denatured protein sample and by limiting staining time to 1.5 h at 13 V/cm.
Park
K B
KB
Department of Food Science, University of Massachusetts, Amherst 01003.
Labbé
R G
RG
D015153:Blotting, Western / Q000379:methods; D003102:Colloids; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D006046:Gold; D006358:Hot Temperature; D007460:Iodoacetamide; D011489:Protein Denaturation; D011506:Proteins / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet; D013194:Staining and Labeling; D001616:beta-Galactosidase / Q000032:analysis
D003102:Colloids; D011506:Proteins; D006046:Gold; D001616:beta-Galactosidase; D004229:Dithiothreitol; D007460:Iodoacetamide