Being able to trust the experimental data is critical for experimental research. This is certainly also true for Western Blotting as one of the most commonly used methods in cell biology laboratories due to its high specificity and sensitivity.
Hence there is a critical need for comprehensive reporting of Western Blotting conditions to improve the accuracy and reproducibility of data based on this technique.
To address this need, Jennifer Gilda and colleagues have developed the Western blotting minimal reporting standard (WBMRS), which covers 10 critical points:
- Information regarding the primary and secondary antibodies used.
- Molecular mass of band of interest should be shown on the blot.
- The amount of total protein loaded onto the gel.
- Type, amount and extent of use of blocking reagent.
- Washing solution used, how often and for how long.
- Amount and incubation time of primary antibody (incl. primary antibody buffer).
- Amount and incubation time of secondary antibodies (incl. secondary antibody buffer).
- How the image was collected.
- The reagent used for detection and membrane incubation time.
- The software used for signal strength quantification (if required).
These are certainly important criteria to support scientist reproducing reported experiments, but what is missing here is a clear guide how to present results obtained:
Over the years, it has become common practice to crop representative blots to reduce their overall size and to save journal space. Removal of unnecessary clutter may indeed help focus the eye of the reader and to improve clarity and readability of the scientific article.
However, quite often, a blot is cut into a narrow strip or cropped around individual bands to remove ugly (beautification), unexplainable or confusing areas.
Not only can it mislead the editor, referee, and ultimately the reader, but it can also hide important information, potentially pointing to real biological insight like in Figure 1 (e.g. “What is the origin of the band below 80 kDa as the band intensity is inversely proportional to the band around 100 kDa?”).
It may also lead to a false impression of antibody specificity. Especially for Figure 2, it is not easily understandable why the space available around the EZH2 signal was not better used – leaving the reader with the question whether or not important information has been removed…
In addition, the red arrow points towards a visible vertical line between lane 2 and 3, which normally represents some unnatural appearances and image manipulation and should be treated with caution. If splicing different gels together is unavoidable, a black line or gap should be left so that it is clear that the parts had separate origins.
Regarding the ability to compare different samples and signal intensities, it is important that these signals come from the same gel/blot as the exact same blotting conditions cannot be guaranteed. Thus, mixing and matching bands from various gels (as shown in Figure 3) is meaningless if the purpose of the experiment was to compare bands between different samples and treatment conditions.
These examples show that it is critical to think about the best approach how to present data generated by SDS-PAGE and Western Blotting. For every adjustment and manipulation (e.g. cutting, splicing) that is done, it has to be ensured that the resulting image still represents an accurate representation of the original data!
As exemplified by Figure 3, we can only effectively utilize Western blotting data if the way we analyse the Western Blotting results obtained is fit-for-purpose and qualified to reach the conclusions presented in the paper.