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How Western Blotting Works

Problem: Nearly 40 years after its introduction, western blotting continues to be a powerful method for protein quantitation. Arguably, finding the right antibody to detect the protein of interest is the technique’s most critical and challenging step.

by Bio-Rad Laboratories
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Figure 1. Validation data for two PrecisionAb candidates. A) This carbonic anhydrase IX (CA9) mouse monoclonal antibody failed validation because it exhibits nonspecific binding and low signal-to-noise ratio. B) This cyclin-dependant kinase 2 (CDK2) mouse monoclonal antibody passed validation showing high specificity and sensitivity

A recent Bio-Rad survey found nearly 60 percent of researchers blamed the primary antibody when their immunoblots failed. Even if the antibody does work, it may bind to other proteins in addition to the target protein or may not be sensitive enough to detect low abundance targets, complicating analysis of the results. Scientists using non-specific antibodies have gone on to unknowingly publish erroneous data. One reason this occurs is because vendors provide insufficient validation information, leaving researchers in the dark about the antibody’s true binding efficiency and overall performance. Furthermore, manufacturing and quality control standards can be lax, which means there is no guarantee that buying different lots of the same antibody from the same supplier will produce the same results. 

Solution: Vendors cannot realistically validate antibodies for every possible application and sample treatment or type. Validating for a broad range of antibody applications would be prohibitively expensive, driving up product prices. An alternative approach to ensure reliability is for vendors to narrow antibody selections. Bio-Rad, for example, is pioneering this approach with the introduction of its PrecisionAb™ Antibody product line, specifically validated for western blots. The new line is a targeted approach to overcoming many of the aforementioned industry challenges.

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“As far as lot-to-lot variability is concerned, we’re testing every lot that we bring in,” says Mark Shulewitz, a senior scientist in the Content Business Development Team at Bio-Rad. “A new lot has to work or we don’t accept it.”

Additionally, while some vendors rely on over-expressing the target protein or concentrate the sample (preparing microsomes or nuclear fractions, for example) to show antibody binding, each PrecisionAb antibody is selected based on highly sensitive and specific detection of endogenous levels of target proteins without any special sample enrichment (Figure 1).

A second solution for improved reliability comes from greater transparency and information. Bio-Rad, for instance, has committed to presenting the full western blot image, including negative and low signals, to give a better indication of an antibody’s overall performance across 12 different cell lines. This information is critical for investigators because a low or negative result “doesn't mean the protein isn’t there, it's just—relatively speaking—much less abundant,” Shulewitz explains.

To facilitate optimization and in-house validation by the researcher, the complete protocol used for the validation, along with a positive control lysate, is available for each PrecisionAb. This provides researchers increased confidence in assigning bands, as they can directly compare their western blots with the vendor’s validation data— which uses the same protocol and positive control lysate. As a final move to empower researchers, trial sizes are available for all PrecisionAb antibody targets. This allows scientists to “try before they buy,” experimenting with their own samples and study environments to confirm the product works before committing to a full vial.

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