We encountered β-mercaptoethanol-dependent artifact signals in western blot analyses using polyclonal antisera. Replacing β-mercaptoethanol with dithiothreitol in the loading buffer did not eliminate the artifact signals. However, lowering the concentration of either dithiothreitol or β-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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology