Western Blotting Troubleshooting Tips

Although western blotting is a relatively simple technique, it creates a surprising amount of work for those who provide technical support. High background, poor signal, or the presence of “too many bands” are among the most common complaints, with many researchers assuming these to be the fault of primary antibody reagents. Here, four western blotting experts explain what else could be to blame, and share tips for improving data quality.

Common complaints

Most antibody datasheets include an image of western blot data, showing one or more clear bands at the expected molecular weight. But when in-house data bears little resemblance to this, researchers often assume that they’ve purchased a "bad" antibody and feel compelled to contact the manufacturer for support. “The complaints I most frequently see are too many bands or bands of the wrong size, followed by weak or no signal for the target protein,” reports Rebecca Northeast, Ph.D., Technical Specialist at Proteintech Group. This point is echoed by Tobias Polifke, Co-founder and Managing Director at CANDOR Bioscience, who comments that high background and a “smear” of bands are other common grievances. Fortunately, issues such as these can usually be addressed by taking a closer look at the western blotting protocol.

Unfavorable consequences

Because western blotting underpins many different research programs, it is critical that data is interpreted correctly. “If you have a lot of off-target bands that you dismiss as non-specific binding, you might be missing key information such as a disease-specific protein fragment or mutation, or a post-translational modification (PTM) associated only with certain tissues,” explains Northeast. “Conversely, by wrongly assuming a particular band to be the protein of interest, your research could be set back as you pursue leads based on incorrect data.” Open-access resources such as PAXdb and UniProt offer valuable insights into protein abundance and known isomers/PTMs/potential complexes, respectively, and can be of great help in making sense of results.

Simple solutions

“One of the first steps toward producing reliable western blot data is selecting the right primary antibody,” notes Joanna Porankiewicz-Asplund, Ph.D., Technical Support Manager at Agrisera. “This should be validated for the western blot application (each technique requires separate validation) and proven to detect the target in the chosen species and sample type (e.g., a specific organ or cellular fraction). Where species reactivity has not been confirmed by the antibody manufacturer, a common mistake made by researchers is to blast the whole protein sequence between species and assume that high homology is enough to ensure the antibody will work. Yet, because antibodies are often raised against short peptides or partial proteins rather than the full-length biomolecule, this strategy can be misleading. By paying closer attention to how the antibody was produced, many unnecessary experiments could be avoided. In turn, this information can help determine other techniques in which the antibody may potentially be used; for example, antibodies that recognize domains buried in the 3D structure of a protein will be unsuitable for techniques like immunoprecipitation, where antibodies binding to exposed regions should be chosen.”

Sample preparation is equally important, especially when you consider that “too many bands” can frequently be attributed to sample degradation. It is recommended that fresh lysates are used wherever possible and that these are prepared on ice using pre-chilled reagents and equipment. Other ways of minimizing sample degradation include adding protease inhibitors to lysis buffers, and boiling samples immediately after extraction. “One point to note is that some membrane proteins may aggregate upon heating to form insoluble particles,” cautions Northeast. “Thus, heating should be avoided here. Moreover, because 'too many bands' may also occur due to under-loading (which favors non-specific antibody binding), it is essential to check the protein concentration of samples to ensure sufficient material is being loaded on to the gel.”

Once samples have been properly prepared and potential antibody reagents identified, it’s time to run the western blot. At this point, it’s sensible to use the antibody manufacturer’s recommended protocol as a starting point from which staining can be optimized in-house. Anne Sloan, Ph.D., Technical Support Scientist at Cell Sciences, suggests running alternate lanes of a molecular weight ladder alongside positive and negative control samples at several concentrations before cutting the membrane into strips to assess different assay parameters. “Optimization should include testing various concentrations of primary and secondary antibodies, and fine-tuning blocking and washing steps,” she says. “It is also important to choose a suitable visualization method; for example deciding between fluorescent or chemiluminescent detection, and determining whether additional signal amplification is required.”

Another factor to consider is the membrane. Nitrocellulose and PVDF membranes are most commonly used for western blotting, but researchers may be unaware that these bind proteins via distinct mechanisms. “Nitrocellulose membranes are negatively charged and exert their binding effects through electrostatic interactions with positively charged regions on proteins,” says Northeast. “In certain cases, these interactions can mask antibody binding sites, resulting in poor signal. In contrast, PVDF membranes bind proteins via hydrophobic interactions. Most antibodies are targeted to hydrophilic domains on proteins and PVDF membranes leave these regions free. The take-home message here is that switching from one membrane type to another can have a big impact on improving target signal.”

Blocking should also be carefully optimized, including the choice of reagent. Although bovine serum albumin (BSA) and milk powder are widely used for blocking (and for diluting antibody reagents), both have their drawbacks. “BSA is not a very effective blocker for membranes due to its high molecular weight,” explains Polifke. “To get a dense layer on a surface, it is better to use a broad spectrum of differently sized molecules such that you achieve faster and more complete coverage. Milk powder goes some way toward addressing this since it contains multiple protein types; however, it suffers from extreme batch-to-batch inconsistency. For faster, more reliable blocking, researchers should consider using a reagent such as The Blocking Solution, which comprises fragmented, chemically-modified casein.”

Tips for success

In addition to the points already mentioned, there are many other steps researchers can take to ensure western blotting success. The following list combines the advice provided by all four contributors to this editorial.

• Understand your target—refer to sites such as PAXdb, UniProt, and the Bio-Analytic Resource for Plant Biology for information about protein abundance and known isomers/PTMs/potential complexes; mine the literature to see how others have detected your chosen protein (check that publications include a reference to the antibody product number, name, and supplier); antibody datasheets are valuable sources of information when provided by reliable suppliers
• Take care during sample preparation—use fresh lysates wherever possible; prepare samples on ice using pre-chilled reagents and equipment; include protease inhibitors in lysis buffers; boil samples immediately after extraction where appropriate (Agrisera recommends heating at 75^oC rather than at 95 or 100^oC for 5 minutes); always check protein concentrations
• Ensure correct conditions are used for running the gel and performing the transfer—mis-shaped bands can result from bubbles being present between the gel and the membrane; "smiling" bands may be due to the gel overheating or being run too fast; blurry bands may be a product of too high a voltage
• Use properly validated antibody reagents from a reliable source—check antibody datasheets for validation data; be sure to store antibodies correctly, avoiding freeze-thawing
• Always include appropriate controls—use resources such as PAXdb and the Human Protein Atlas to identify relevant positive and negative sample types; consider performing knockdown or knockout studies using RNAi or CRISPR technology; omit primary antibody reagents to check secondary antibodies are not showing non-specific binding; check to see whether the antibody manufacturer has generated these data
• Refer to the antibody manufacturer’s recommended protocol for guidance but always optimize assay conditions in-house—titrate primary and secondary antibody reagents; when working with animal material, consider using secondary antibodies that have been pre-adsorbed against the sample species; investigate the effects of incubation time/temperature; evaluate different wash buffers; if using ECL detection, select a reagent with appropriate sensitivity
• Try replacing BSA or milk powder with more modern reagents—LowCross-Buffer^® minimizes cross-reactivities, matrix effects, and non-specific binding, while ReadyTector^® enables blocking and both primary and secondary antibody incubations to be performed in a single step
• Consider switching a nitrocellulose membrane for PVDF, or vice versa; select a pore size of 0.25 µm for proteins <25 kDa and 0.45 µm for proteins >45 kDa
• Think about protocol reduction—try using conjugated primary antibodies such as CoraLite^® primaries to streamline workflows and decrease the number of places where things might go wrong; consider moving from ECL to fluorescent detection to avoid the need for stripping and re-probing; design workflows to allow for using ECL and fluorescent detection interchangeably

Lastly, never be afraid to ask for help! Antibody manufacturers and their technical support teams can provide a wealth of advice, based on having seen many common western blotting problems before, and can help researchers to achieve publication-quality western blot data first time.