A systematic approach to quantitative Western blot analysis

Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process. •We provide an updated view on several critical concepts of quantitative Western blotting including ratiometric analysis, normalization, validation of controls, antibodies, and detection of combined linear range.•This review provides a systematic workflow to gather quantifiable and reproducible data via Western blot.•Relevant to researchers intending to use Western blot technology, in basic sciences and translational biomedical research.