Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents.
We encountered beta-mercaptoethanol-dependent artifact signals in western blot analyses using polyclonal antisera. Replacing beta-mercaptoethanol with dithiothreitol in the loading buffer did not eliminate the artifact signals. However, lowering the concentration of either dithiothreitol or beta-mercaptoethanol eliminated the background problems and allowed specific detection of the target protein. These results are consistent with the background signal being caused by anti-keratin antibodies in the antisera and keratin contamination of reagents. This study highlights the importance of testing a range of reducing agent concentrations when trying to eliminate artifact bands from western blots. However, this method may not be applicable when target proteins have disulfide bridges.
Lee
T-F
TF
Plant Pathology Department, 211 Buckhout Laboratory, The Pennsylvania State University, University Park, PA 16802, USA.
McNellis
T W
TW
D016477:Artifacts; D015153:Blotting, Western; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D007633:Keratins / Q000032:analysis* / Q000737:chemistry; D019163:Reducing Agents / Q000737:chemistry*
D004220:Disulfides; D019163:Reducing Agents; D007633:Keratins