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

Abstract

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

Financial & competing interests disclosure

This work was supported by National Institutes of Health Grants HL096819 and HL080101 and UC Davis research funds. AV Gomes presented a talk on ‘Can Western blots be trusted?’ at the Experimental Biology 2014 meeting which was sponsored by Bio-Rad. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key issues

• Signal detection. The ease at which an x-ray film becomes saturated with signal gives investigators a false sense that film is more sensitive than digital imagers. Laboratories should be encouraged to switch from x-ray films to digital imagers or learn how to validate that signals are not saturated on x-ray films.

• The misconception that housekeeping proteins are the best normalization method for western blots needs to be addressed.

• The need for researchers to load sample amounts in which target detected by the antibody is within the linear range.

• High volume of poor quality antibodies available.

• Need for positive and negative controls to validate antibodies.

• Need for determining and stating the molecular weight of the target protein on blots.

• Need for an unbiased database allowing researchers to document good- and poor quality antibodies.

• Need for publications to describe the western blotting technique utilized in more detail. A minimum reporting standard for western blotting data should be established.

• Need for recommended procedures for protein quantification.

Notes