For decades, western blotting has been used to provide a simple yes/no answer regarding protein expression in a sample. But with every step in the process seeing improvements, this tried and trusted technique is now far more than just a qualitative method for visualizing protein bands on film. Here, we review recent advances in western blotting and discuss how these are providing faster, more accurate results.
Novel gel technologies
Following sample preparation, western blotting begins with protein separation on a gel. Pre-cast gels have long been used as a convenient alternative to hand-poured gels, providing rapid run times and greater experimental consistency. However, the addition of stain-free technology has opened up new capabilities. “Stain-free gels contain a trihalo compound that produces a fluorescent product when crosslinked to tryptophan residues, with crosslinking achieved by briefly exposing the gel to UV light after electrophoresis,” explains Paul Liu, PhD, product manager, western blotting, imaging, and software at Bio-Rad. “This allows for rapid detection of protein bands in the gel using an imaging system and, because the crosslinked products can also be detected on the membrane after protein transfer, it streamlines the process of monitoring transfer efficiencies.” Stain-free technology can also be used for total protein normalization.
More relevant protein normalization
According to Jeff Harford, senior product marketing manager at LI-COR, total protein normalization has become a preferred approach to using housekeeping proteins (HKPs) for normalizing western blot data. “Studies have shown that HKPs are often influenced by drug treatments,” he says, “leading to inaccuracies in data analysis. Additionally, it can be difficult to determine a combined linear range for the HKP and the protein of interest since HKPs are frequently overexpressed. Because the total protein population is less susceptible to drug-induced change compared to a single HKP, and since total protein stains have linearity over a broader range than most HKPs, total protein normalization is now widely recognized as being more relevant. This awareness continues to grow thanks to publishers providing clear guidelines and best practices to authors.”
Faster protein transfer
By significantly reducing the time taken for protein transfer, semi-dry systems have already seen huge uptake. More recently, they have been complemented by rapid semi-dry systems such as Bio-Rad’s Trans Blot Turbo, which allows for protein transfer in as little as three minutes when used with the company’s TGX gels. “Modern, rapid blotting devices have proven hugely successful in providing complete protein transfer without compromising data quality,” notes Dr. Tobias Polifke, co-founder and managing director at CANDOR. “The scientific community has been quick to embrace these changes, mainly due to the fact that speed is almost the only factor to consider in switching from wet blotting to an alternative method. In contrast, changes to the western blotting biochemistry remain far slower to be adopted, even though these can offer many other benefits in addition to shorter workflows.”
More reliable immunostaining
Improvements to the immunostaining components of western blotting have been many and varied. Of these, it is arguably antibody quality that has received the most attention, with many suppliers investing considerable time and effort to improve antibody validation in recent years. Yet despite both primary and secondary antibody reagents becoming increasingly more reliable, their binding potential is often overshadowed by the use of inappropriate ancillary reagents. Expanding on his point about slow adoption of improvements to the western blotting biochemistry, Polifke highlights researchers’ reluctance to move away from using reagents such as milk powder and BSA for blocking and antibody dilution.
“Milk powder is an ill-defined foodstuff with extreme lot-to-lot inconsistencies—perfect for the coffee mug but a poor choice for immunodetection,” says Polifke. “BSA for research use is subject to more rigorous quality control, but there are far better options now available. These include a solution that provides blocking in just two minutes, with low background for even low affinity antibodies, and specialized affinity-discriminating antibody diluents that minimize unwanted interference.” CANDOR has also simplified the immunodetection workflow and reduced it to as little as 45 minutes by developing a solution (ReadyTector®) enabling blocking, primary antibody incubation, and secondary antibody incubation to be performed in one step.
Image: Western blot detection using standard sequential protocol with multiple buffer changes (left) and using ReadyTector® (right). Image provided by CANDOR.
Quantitative multiplex detection
Another area where change has been relatively slow to take effect is the switch from chemiluminescence and film for western blot detection to fluorescence and digital imaging. “The perception that using chemiluminescence and film provides the most sensitive detection has meant many researchers are reluctant to switch from using existing methods,” reports Harford. “However, in our experience, the sensitivity of infrared western blot detection now rivals that of chemiluminescence and film, a factor that has contributed to fluorescence and digital imaging becoming a preferred option.” Liu adds that a major advantage of fluorescent detection is that it provides the ability to multiplex. “In order to detect multiple targets using chemiluminescent detection, researchers have historically stripped and re-probed the same membrane or cut each blot into sections,” he says. “With fluorescent multiplexing, a single, publication-ready image captures all the targets, each in its own color channel, and these can then be accurately quantitated in parallel to greatly increase the value of western blot data.”
More convenient reporting
With the vast majority of researchers now using digital imagers to capture western blot data, many film manufacturers have discontinued production. In turn, this has led those manufacturing digital imaging systems to develop better methods for data analysis. While users are currently accustomed to manually defining the lanes and identifying the bands on each blot image, this approach is notoriously prone to user-bias. “Traditional western blot analysis software was not designed to incorporate the best practices published by journals such as JBC,” says Harford. “As such, working through the necessary steps to get western blot data analyzed could be quite tedious. By designing a software with best practices as the foundation, we systematically walk researchers through linear range validation, normalization, replicate analysis, and other essential data handling steps, bringing the CV variation from person to person down to just 3% compared to greater than 25% with traditional software.”
Although it may seem that there is little room for further improvement to western blotting, a challenge remains in raising awareness of the benefits that can be gained by switching to modern technologies. Western blotting remains your author’s favorite laboratory technique, so rest assured that a close eye will be kept on any future developments.