Western blot is one of the most reliable methods to characterize protein expression. Using straightforward protocols and relatively inexpensive reagents, it provides a semi-quantitative visual assessment of even low-abundance targets. This is achieved through partial protein purification according to size, prior to highly specific antibody-based detection of the target of interest. Western blot has been improved with innovative detection methods, more sensitive imaging technologies, and a variety of automated instrumentation. More recently it has evolved into a sophisticated gel-free, blot-free technique. With many researchers generating western blot data to support experimental results, and most antibody manufacturers using western blot to validate their products, this trusted method remains extremely popular. Understanding the different options, how they work, and the benefits they offer can increase the chances of western blot success.

Western blot delivers key insight

“A major advantage of western blot is that it allows researchers to observe protein isoforms, which are reflected in multiple bands that display the size differences,” reports Karolina Szczesna, senior product manager and technical support at Proteintech Group. “Although immunocytochemical staining and IHC produce magnificent imaging, they cannot really distinguish protein isoforms, making western blot useful to confirm experimental findings.”

“Western blot also allows the detection of protein modifications such as phosphorylation, ubiquitinylation, glycosylation, methylation, and SUMOylation,” adds Russ Yukhananov, CEO at Precision Biosystems. “These often result from a complex interplay of genetic, epigenetic, and environmental factors, and cannot be deduced from genomic information alone. Western blot is therefore an important tool to investigate the role of specific proteins within cellular pathways, disease pathogenesis, and organism development.”

Moving away from the dark room

“Western blot has evolved considerably over the years with the introduction of fluorescence and the ability to multiplex, while improvements to chemiluminescent reagents have made it possible to detect even femtogram amounts of protein,” notes Lindsey Kirby, product manager at Syngene. “Using gel imaging systems such as our Syngene range, researchers can develop chemiluminescent or fluorescent blots at the lab bench, eliminating the need to spend extended periods of time in the dark room. A further advantage of the Syngene systems is that many of them are compatible with stain-free technology—a faster and more convenient alternative to Coomassie staining, which has made it easier to normalize western blot data without the worry of whether you have used the correct housekeeping protein.”

“Other important developments within the field of traditional western blotting include stronger gels, enhanced transfer buffers, and the incorporation of directly labeled primary antibodies into immunostaining protocols,” adds Shuoya Tang, marketing manager at Expedeon. “Our RunBlue™ gels are up to ten times more robust than conventional hand-poured gels, while our InstantBlot™ transfer buffer is designed to reduce wet transfer times to as little as ten minutes (where less than 100 kDa) without the excessive heat generation that may denature proteins.” Complementing these products, Expedeon’s Lightning-Link® kits allow researchers to directly label their primary antibody, thus eliminating the need for secondaries and reducing time and wash steps needed for western blotting.

According to Szczesna, better validated antibody reagents have greatly increased researchers’ confidence in western blot data. “Nowadays, western blot has the capacity to be more complex due to the availability of highly optimized antibodies against a broad range of targets,” she says. “Proteintech antibodies are raised against whole proteins, an approach that delivers superior antibody binding to the native target at multiple epitopes compared to peptide immunogens. This results in greater specificity and sensitivity, and lower background within western blot and other immunoassays.”

Gerry O’Beirne, global product manager at GE Healthcare, explains that western blot tools and techniques have evolved over time to meet the needs of researchers. “Proteomics kicked off the ‘omics’ discovery era as the route to identify proteins as potential therapeutic targets,” he says. “Western blot has contributed significantly, with improvements being seen in both one-dimensional and two-dimensional protein separation; methodologies to extract target proteins for further investigation; the development of new imaging platforms; and the evolution of readouts from radiochemical detection to ECL to fluorescence. More recently, near-infrared (NIR) detection has become possible, as exemplified by GE Healthcare’s Typhoon NIR Plus imaging platform.”

Automated western blot processors improve reproducibility

“The major limitation of traditional western blot was once the lack of reliable automation, especially for the manual processing of immunodetection,” reports Yukhananov. “Precision Biosystems’ BlotCycler™ and similar instrumentation have filled this gap, minimizing operator error to deliver improved reproducibility. Since automated systems remove bias and free up considerable amounts of time, there seems little doubt that they will eventually replace manual alternatives.”

Szczesna agrees, noting that while automated immunodetection systems are becoming a standard in the industrial sector, their use remains rare in academia. “Automated systems are used routinely for screening and drug development programs,” she says. “Within academic research, it is more common to run a small sample number with different sets of antibodies, meaning that automated systems are less advantageous. Some academics prefer to follow traditional methods and established in-house protocols and have little requirement for high-throughput screening.”

Gel-free systems are a modern alternative to traditional western blot

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Gel-free systems are one of the most exciting innovations in western blot technology, affording the benefits of greater reproducibility, higher throughput, shorter assay time, and the capacity to generate true quantitative data. “By providing complete automation of the entire protein separation and detection process through capillary electrophoresis, Simple Western has propelled the western blot into the 21st century,” says Steven Le, product marketing manager for Simple Western at ProteinSimple, a Bio-Techne brand. “Simple Western instruments are designed to provide scientists with the greatest possible number of tools and utilities to thoroughly characterize proteins.”

Image: Comparative total protein data and the expression of the housekeeping protein β-actin in six human whole tissue lysates loaded on an equal protein basis (0.3 mg/mL). Assay performed on Jess. Image courtesy of Bio-Techne.

Le explains that researchers can characterize proteins by size or charge via Simple Western assays on the Peggy Sue system. “Alternatively, using Jess, it is possible to perform an immunoassay across chemiluminescence, NIR, and IR detection channels simultaneously, while also running a total protein assay within the same capillary,” he says. “Detection of a low-abundance protein on the chemiluminescence channel and a high-abundance protein on the NIR channel is a unique form of multiplexing and is only possible on the Simple Western platform.”

Yukhananov points out that while gel-free systems are extremely useful to screen the effects of large numbers of compounds upon protein expression, many antibodies have not yet been tested with this technology. ProteinSimple has addressed this through an ongoing antibody validation program, with a wide range of antibodies demonstrating successful performance in the Simple Western application. “Soon enough, I think these systems will very much become the mainstream, especially in labs that do a lot of HTS and biomarker analysis,” says Szczesna. “They may also overtake traditional western blotting within academia, where timing can be crucial to the publication of data.”