The necessity of and strategies for improving confidence in the accuracy of western blots

Expert Rev Proteomics. 2014 Oct;11(5):549-60. doi: 10.1586/14789450.2014.939635. Epub 2014 Jul 25.


Western blotting is one of the most commonly used laboratory techniques for identifying proteins and semi-quantifying protein amounts; however, several recent findings suggest that western blots may not be as reliable as previously assumed. This is not surprising since many labs are unaware of the limitations of western blotting. In this manuscript, we review essential strategies for improving confidence in the accuracy of western blots. These strategies include selecting the best normalization standard, proper sample preparation, determining the linear range for antibodies and protein stains relevant to the sample of interest, confirming the quality of the primary antibody, preventing signal saturation and accurately quantifying the signal intensity of the target protein. Although western blotting is a powerful and indispensable scientific technique that can be used to accurately quantify relative protein levels, it is necessary that proper experimental techniques and strategies are employed.

Keywords: housekeeping protein; immunoblot; loading control; quantification; stain-free gel; total protein normalization; western blotting; western blotting accuracy; western blotting strategy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Antibodies / chemistry
  • Blotting, Western / methods
  • Blotting, Western / standards*
  • Proteins / analysis
  • Proteins / standards
  • Reference Standards
  • Staining and Labeling


  • Antibodies
  • Proteins