Thus, editors and reviewers of scientific journals are looking at western blot results, particularly at the densitometric analysis to determine the fold differences in protein expression, with greater scepticism, often requesting the raw data files.Ĭhemiluminescent western blot data, derived from film-based detection, poses distinct challenges in producing quantifiable, reproducible data. This underlines the negative perception by which the scientific community views the western blot data. A recent report predicts that approximately 25 % of the accepted papers include at least one inappropriately manipulated figure and many of these are associated with western blotting. These include challenges related to every step of the western blotting procedure, from sample preparation, normalization, SDS–PAGE gel loading, protein transfer, primary and secondary antibody selection, incubations, and washes, detection method selection to densitometric analysis. However, there are many potential stumbling blocks in this procedure that can preclude reliable results. This multistep method determines the presence or absence, size, and modification or degradation states of target proteins, as well enables the quantitation of proteins from complex protein mixtures or homogenates. Western blotting has been a staple in life science labs for several decades-ever since researchers published the first detailed description of this protein detection technique in 1979.

Imagej quantification of western blot