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

T. F. Lee, T. W. McNellis

Research output: Contribution to journalArticle

5 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)141-143
Number of pages3
JournalAnalytical Biochemistry
Volume382
Issue number2
DOIs
StatePublished - Nov 15 2008

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Mercaptoethanol
Reducing Agents
Keratins
Artifacts
Dithiothreitol
Western Blotting
Immune Sera
Disulfides
Anti-Idiotypic Antibodies
Buffers
Proteins
Contamination
Antibodies
Testing

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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Elimination of keratin artifact bands from western blots by using low concentrations of reducing agents. / Lee, T. F.; McNellis, T. W.

In: Analytical Biochemistry, Vol. 382, No. 2, 15.11.2008, p. 141-143.

Research output: Contribution to journalArticle

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