With western blot data facing increased scrutiny from publishers, researchers need to have confidence in their results. This editorial offers practical guidance for ensuring that your western blots are both accurate and reproducible.
A technique with enduring popularity
Western blotting is powerful laboratory technique that produces straightforward yet highly informative data. “With western blotting, you can characterize so much about an individual protein,” says Afrida Rahman-Enyart, Ph.D., Product Manager and Scientific Liaison at Proteintech. “For example, you can observe its presence, abundance, and mass, as well as identify post-translational modifications. It is also possible, with the combination of immunoprecipitation, to detect protein-protein interactions.”
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“When designed correctly, western blots can provide semi-quantitative data and allow comparisons of the amounts of target protein present in different samples,” adds Jade Fee, Application Scientist at Azure Biosystems. “Despite many other techniques having been developed since western blotting was first reported over 40 years ago, researchers and publishers prefer seeing western blot data because one experiment can yield types of information that other single analytical methods cannot provide.”
Best practice recommendations
Although western blotting is relatively simple to perform, it is important that researchers do not become complacent when designing and running experiments. The following best practice recommendations will help you to keep your western blots on track.
Selecting the right lysis buffer to solubilize the target of interest is a critical step during sample preparation. “Although RIPA buffer is the gold standard, it is not compatible with all proteins, cells, and tissue types,” reports Rahman-Enyart. “For example, RIPA buffer’s harsh properties make it ideal for hard-to-solubilize targets such as nuclear and membrane-bound proteins, yet a Tris-HCl buffer might be more advantageous for solubilizing cytoplasmic proteins. It is recommended to test several different buffers with varying concentrations of chaotropic agents/detergents, and to always add a protease inhibitor cocktail to prevent target degradation.”
Following solubilization, SDS sample buffer is typically added to lysates prior to boiling for 5–10 minutes to enable protein denaturation. However, boiling can cause membrane proteins to aggregate, resulting in a high molecular weight and an inability to move through the gel. “When dealing with membrane proteins, you might need to lower the temperature when heating or add chaotropic agents to aid protein solubilization,” suggests Rahman-Enyart. Another best practice recommendation is to ensure that protein concentrations are consistent between samples when comparing different experimental conditions. The Bradford assay is the most widely used method for protein quantification.
- Gel electrophoresis and protein transfer
The molecular weight of the protein of interest is the main factor to consider for gel selection. In general, larger proteins require lower percentage gels, and vice versa. You should also pay attention to the number of wells per gel, making sure there are enough wells available for all of your samples plus your controls and molecular weight marker.
When it comes to membrane selection, researchers face a choice between polyvinylidene difluoride (PVDF) and nitrocellulose. “PVDF membranes are more durable, making them ideal for stripping and re-probing,” explains Rahman-Enyart. “They also tend to work well for low expressed proteins and antigens that are hydrophilic. Nitrocellulose membranes have a high protein-binding capacity and are often preferred for hydrophobic antigens, although they require more careful handling than PVDF as they can be brittle and tear easily.”
Once a suitable gel and membrane have been identified, correctly assembling the transfer sandwich is key. According to Fee, a complete and even protein transfer is fundamental to a successful western blot. She cautions researchers to remove any air bubbles after assembling the transfer sandwich, and to provide even wetting across the entire membrane. “Also, use a reversible total protein stain to confirm successful transfer before continuing with the blocking and antibody incubation steps,” she advises.
There are numerous factors to consider for immunostaining, not least selecting the right antibody reagents. “The antibodies that you use for western blotting should be specific to your target of interest and should not cross-react with any other proteins,” says Rahman-Enyart. “A dirty antibody will give a dirty blot, so review the validation data from the supplier closely and be sure to perform specificity tests using positive and negative controls.”
Another tip is to be consistent with the antibodies used, particularly the primary antibody. Ideally, in an experiment that involves multiple blots, a single preparation of the primary antibody will be aliquoted out into different tubes and stored so that an identical antibody preparation can be used for each blot. This avoids repeated freeze-thaw cycles. “Keep in mind that to obtain quantitative information from a western blot, the primary and secondary antibody concentrations should be optimized so that the protein of interest can be detected without signal saturation,” notes Fee.
When it comes to interpreting western blot data, controls are essential reference points. “A positive control enables easy identification of which band corresponds to the protein of interest and might, for example, be a lysate from transfected cells or a tissue that abundantly expresses the protein,” says Rahman-Enyart. “A negative control instead serves to help identify any nonspecific binding or background, which should be excluded during analysis.”
You will also want to decide how the data will be normalized. According to Fee, total protein normalization (TPN) is often preferable to using an internal control such as a housekeeping protein because many housekeeping proteins are not consistently expressed across different tissue types or experimental conditions. “During TPN, total protein is detected either by staining the protein gel before transfer or staining the membrane after transfer and before blocking,” she says. “Using image analysis software such as AzureSpot Pro, the amount of total protein in a lane is calculated and used to normalize the signal of the band of interest.” For normalization of low-expressed proteins, Fee suggests using TotalStain Q, which has a linear range between 1–50 μg of lysate.
Top ten tips for western blotting success
- Choose the right lysis buffer
- Know when to boil your samples
- Ensure that protein concentrations are consistent between samples
- Select the right gel based on acrylamide percentage and number of wells
- Determine whether a PVDF or nitrocellulose membrane best suits your needs
- Assemble the transfer sandwich correctly—remove any air bubbles, ensure even wetting of the membrane, use a reversible total protein stain for confirming successful transfer
- Take care with antibody selection—review the manufacturer’s validation data and perform your own specificity tests using positive and negative controls
- Optimize primary and secondary antibody concentrations
- Always include appropriate controls
- Consider switching from an internal control (e.g., a housekeeping protein) to total protein normalization