Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents

Abstract

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.