Western blotting has been used for decades to visualize and identify proteins, and it remains an essential technique to support antibody validation efforts. Yet while the western blotting process has been improved and optimized for more accurate, sensitive, quantitative, and rapid data, it can still yield unexpected results. Frequently encountered problems can be avoided with close attention to protocols and by leveraging the many technological advances that have been made in western blotting to minimize the need for troubleshooting.
There are several possible reasons for a western blot to have no bands. These include no antigen expression in the source material, too little antigen loaded on to the gel, and antibody-related effects such as the primary antibody being unsuitable for western blotting or being used at the wrong concentration. Exchanging the source of target protein or concentrating the sample can overcome antigen-based issues, while antibody datasheets provide valuable information about antibody performance.
A lack of bands may also be due to poor technique, for instance if the protein transfer did not work properly. One way to quickly optimize transfer conditions is to use a rapid blotting transfer system, since these can achieve protein transfer in as little as 3 minutes. It is also possible using stain-free imaging to visualize all steps of electrophoresis and blotting, saving time and reagents wasted on western blots with problems that would not otherwise have been detected until the later stages of blot processing and development.
The appearance of bands at a higher molecular weight than expected can often be explained by the presence of post-translational modifications, or by the occurrence of dimers, multimers, or protein-protein interactions due to samples having not been fully reduced and denatured. Suspected modifications can be removed using enzymes, whereas adding fresh reducing agent and reheating samples eliminates multi-protein complexes. Bands occurring at a lower molecular weight are commonly a result of sample degradation; this can be avoided by adding protease inhibitors to the lysis buffer and keeping samples on ice.
Faint bands or weak signal are most often antigen- or antibody-related, as already described, however a low transfer efficiency may also be to blame. Where a rapid blotting transfer system is unavailable to optimize transfer conditions, transfer efficiency can be evaluated by staining the membrane with Ponceau S and/or the gel with Coomassie dye after transfer, and by noting how well any pre-stained molecular weight markers have transferred onto the blot. Although removing Ponceau S adds extra steps to protocols, this stain provides useful insight to aid troubleshooting.
Buffers can be another cause of weak signal, especially where non-fat dry milk is used in the blocking and antibody solutions. Since non-fat dry milk may mask some antigens, many researchers instead prefer to use a universal buffer that is compatible with a wide range of targets, sample types, and detection methods. An additional benefit of universal buffers is that they can significantly reduce blocking times; down to just 5 minutes in some cases compared to 1 hour using non-fat dry milk.
Another way in which universal buffers improve on non-fat dry milk is highlighted by their use to support western blot detection of phosphoproteins. High background is a common problem here and is often due to the reaction of phospho-specific antibodies with casein, a phosphoprotein present in milk. Universal buffers prevent this issue, reducing background without compromising sensitivity.
High background is especially unforgiving of poor technique and can be caused by many easily preventable operator errors. These include allowing the membrane to dry out, performing inadequate washing between antibody incubations, using high incubation temperatures that diminish antibody performance, and choosing PVDF membrane rather than nitrocellulose. Where the blot is developed by exposure to film, overlong exposures can also lead to unwanted background.
Although film has long been used for western blotting due to its sensitivity—almost any signal can be captured with a sufficiently long exposure—it comes with significant drawbacks. Not only does film have a limited dynamic range, it also adds time and cost to western blot protocols and provides qualitative rather than quantitative data. In contrast, the use of digital imaging systems for western blotting offers researchers a wealth of advantages: a broad dynamic range (≥4 logs) for accurate quantitation of a range of signal intensities, no costly consumables, and digital documentation for a more traceable data trail.
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