Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents |
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Authors: | Lee T-F McNellis T W |
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Affiliation: | Plant Pathology Department, 211 Buckhout Laboratory, The Pennsylvania State University, University Park, PA 16802, USA |
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Abstract: | 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. |
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