![]() “To avoid this, it is best to start with the manufacturer’s recommended dilutions for the primary antibody and follow the secondary antibody dilution guidelines in conjunction with a high-quality substrate. “Weak or no signal is most often seen when antibody dilution and substrate conditions are not fully optimized, and can be further exacerbated if sub-optimal exposure times are used during imaging,” comments Halbrader. We’ve suggested ways of addressing these issues here, and you can also find further help in our Western Blot Troubleshooting Guide. ![]() Western blot problems generally fall into one of several main categories, of which weak signal, unexpected bands, and uniform high background are among the most common. “Taking the time to carefully plan each step of your western blot will help prevent wasted resource and sample material as experiments are repeated.” Troubleshooting “While the authors describe great improvements in transfer speed and efficiency, it is worth noting that a quick high-voltage transfer is not always the answer, especially for larger proteins,” says Anderson. This includes a comparison of different transfer methods whose use, like any stage of the western blot workflow, should always be considered with respect to the target being investigated. “Imaging systems are now available that promote following western blot best practices-such as normalization against a total protein stain rather than a housekeeping protein-meaning data is far more likely to meet the increasingly rigorous standards required for publication.”įor an in-depth overview of emerging techniques for western blot, Cali Anderson, Technical Specialist at Proteintech Group, recommends a recent Future Journal of Pharmaceutical Sciences paper. “ Modern digital imaging has increased the sensitivity and dynamic range for western blot detection, moving us from the qualitative assay that western blots used to be toward the quantitative assay that western blots can be today,” she notes. Search Now Search our directory to find the right western blot products for your research needs.Īccording to Joy De Torres, Applications Scientist at Azure Biosystems, advancements in digital imaging technology have been the biggest factor in changing the western blot landscape to date. And modern gel documentation and western blot imaging systems can provide faster image capture, higher resolution, and more accurate data analysis.” Newly developed high sensitivity chemiluminescent substrates enable detection down to the attogram level to help with identifying low abundance targets. “For example, innovative precast gel chemistries such as Bis-Tris Bolt provide neutral pH conditions and can be run at faster times with increased sample load volumes for better protein resolution and detection analysis. Manager, Product Management, Biosciences Division at Thermo Fisher Scientific. ![]() “Improvements to western blot across reagents, instrumentation, and software have led to better reproducibility, time to results, and throughput,” reports Emily Halbrader, Sr. But modern western blotting is much improved compared to early forms of the technique. ![]() Western blot improvementsįor over 40 years, western blot has retained its status as one of the most widely used protein detection methods in life science research. Here, we comment on how western blot has evolved, before suggesting ways of addressing common issues. Consequently, the types of problems faced by researchers have stuck around. Although western blot workflows have changed since the technique was first developed, the fundamental principles have remained the same.
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