Membranes were immediately transferred to a blocking buffer for fluorescent western blotting (Rockland), and incubated with a gentle agitation for 1 h at room temperature. Protein gels were blotted using the Trans-Blot Turbo transfer apparatus and PVDF Midi transfer packs (Bio-Rad).

Western Blot Transfer and Total Protein Imaging Gel ImagingĪll gels were imaged using the stain-free application on the ChemiDoc MP (Bio-Rad) imager immediately after the protein separation and prior to western blotting. All sample wells were loaded with 20 μl of the protein mixture with separation using the Criterion Dodeca gel apparatus (Bio-Rad) for 1 h at 200 V. Protein Sample Preparation and SeparationĪ mixture containing a lysate from HeLa cells and purified yeast alcohol dehydrogenase (ADH) (Sigma Aldrich) in Laemmli buffer (Bio-Rad) was used as the starting material for separation on Criterion TGX AnykD Stain-Free gels (Bio-Rad). Based on our findings, we propose a rigorous and simple methodology to produce high quality, reproducible, and quantitative western blot data. Here, we will demonstrate how standardized protein samples, when processed with film versus digital imaging methods and different normalization approaches, produce vastly different results. These new tools and techniques eliminate the limitations associated with film-based detection and meet the journal reviewers’ demands for quantifiable protein expression data. By contrast, the rapid evolution of affordable and highly sensitive gel and blot imaging technologies coupled with new reagents gives researchers the means to produce truly quantitative western blot data-as long as the process is carried out with proper technique, validation, and controls. However, unless the experiments are performed with a deep understanding of these limitations, this method of detection is an approximation at the best and often nonquantitative if used inappropriately. The scientific community has largely ignored these challenges, mostly because of a common misperception that film produces the highest quality data from western blots. These problems stem from a low-dynamic range of detection and the difficulty in accurately determining the limit of detection. 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.

Western blot imagej quantification