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Emerging technologies can help researchers overcome the challenges that can arise at any stage of the western blotting process.

Western blotting (or immunoblotting) is a biochemical technique that provides essential information to characterise proteins in complex samples. The technique is relevant to many medical and scientific fields, so it's no surprise that almost all biochemistry labs around the world use the western blot. These labs make the most of the western blot's relatively cost-effective and simple nature, integrating it into a multitude of studies and research developments.

However, although used widely, the western blot can cause frustration when challenges arise. Things can go wrong at any stage of the process, regardless of how experienced the researchers involved are. The western blot has an apparently random tendency to fail at any of the five stages in the process. Nonetheless, there are new technologies on the horizon, and these technologies can quickly identify failed blots and help researchers streamline the western blotting process.

The life sciences journal BioTechniques has published lots of information on the western blot and its associated challenges. Here, BioTechniques examines the five stages of western blotting and introduces a brand-new technology that helps researchers overcome the challenges that can arise at each of these stages.

The Five Stages of Western Blotting

Western blotting recognises a protein's molecular weight, quantity, and post-translational modifications. It is crucial to monitoring changes in proteins, like expression and modifications.

The first stage of the process is the sample preparation. During this stage, researchers use buffers to lyse the sample cells. This makes the target proteins available in a suspension, unfolding the proteins and giving them a negative charge. Sometimes, researchers need to break the sample down using a process like homogenisation, blending, or sonication.

The next stage is protein separation. During this stage, researchers apply electrophoresis to separate the proteins by their mass charge ratio. Electrophoresis uses an electrical current to force the sample through a gel. This process separates charged molecules according to their physical properties. Researchers might need to separate the proteins by various factors, such as electric charge, isoelectric point, or molecular weight. The most common form of electrophoresis, SDS-PAGE, uses polyacrylamide gel and sodium dodecyl sulphate buffers to separate proteins according to molecular mass.

The third stage is blotting. Here, researchers transfer the separated protein from the gel onto a blotting membrane. This membrane is usually polyvinylidene difluoride (PVDF) or nitrocellulose. The blotting process makes the proteins accessible to antibody detection. Researchers can choose from multiple transfer methods, like diffusion transfer, vacuum blotting transfer, capillary transfer, and the most common choice, electroblotting. This blotting method is quicker and more efficient than many methods and uses an electric current to pull the proteins from the gel to the membrane.

The fourth stage is antibody incubation. At this point, researchers prevent non-specific antibody binding by 'blocking' the membrane with buffers like serum, milk, or highly purified proteins. They then bind a primary antibody to the target protein, wash excess primary antibody off, and introduce a tagged secondary antibody to bind to the primary antibody. This secondary antibody detects the target antigen and provides the readout for imaging.

The final stage is imaging and data analysis. Researchers detect the reporter molecule of the secondary antibody using a film, CCD camera, or scanner. This imaging process allows researchers to analyse the sample, establishing the presence, size, and quantity of the target protein.

Bio-Rad's Stain-Free Western Blotting Technique

Although issues can arise at any of these five stages, new technologies that tell researchers when an issue has arisen are now available. For example, Bio-Rad's stain-free western blotting technique eliminates the need for researchers to stain gels and membranes. The technology confirms that researchers have completed key steps, like electrophoresis and blotting, successfully. This avoids researchers applying expensive antibodies and detection agents onto already failed blots, saving time and money.

The Western Blot's 40^th Birthday

2019 marked the western blot's 40^th birthday, and organisations across the life sciences sector - including BioTechniques - celebrated the success that the technique has brought to the industry in that time. Today, the western blot is a cornerstone of several protein studies. In fact, researchers cite the technique in 9% of all protein-related papers.

Western blotting is a versatile technique that benefits a host of applications, from basic research to high-level diagnoses. The technique is often particularly useful in the early stages of studies. Researchers frequently use it to validate the successful transfection of a cell colony with a non-viral vector. They can use the western blot to identify a specific protein in a sample and determine rudimentary information about its size. That said, the western blot's confirmatory nature can also make the technique useful in the later stages of studies, particularly in confirming the success of gene editing techniques like CRISPR.

About BioTechniques

Since its launch in 1983, BioTechniques has become a go-to publication for scientific and medical practitioners of virtually all disciplines. The journal was the first publication to feature peer-reviewed primary research articles that spotlighted life sciences methodologies and techniques. Today, as a globally trusted journal, BioTechniques publishes the latest information and updates on techniques, methods, and instrumentation in the life sciences sector. The open-access journal distributes this content both in print and online.

Many professionals also make the most of BioTechniques' vast online community, where users access a wealth of webinars, videos, interviews, podcasts, and eBooks. Researchers, industry experts, and budding scientists from all over the world find these resources invaluable. Whether these professionals work in life sciences or find life science applications useful for their technologies, BioTechniques offers the resources they need to develop their professional knowledge.

BioTechniques is part of Future Science Group, the progressive scientific and medical publisher that curates cutting-edge research in its 34 peer-reviewed journals. Other titles include Nanomedicine, Regenerative Medicine, and Future Oncology. To date, Future Science Group has published over 50,000 articles and receives over 5 million article downloads each year.