An Automated Age for Western Blotting

Western blotting has been a core technique in molecular biology for many years. However, due to the nature of the technique, protocols can require laborious optimization and even then, results can be highly variable. When striving for consistency and reproducibility, problems such as these can be a significant barrier in research and advancement.

For a number of years, leading scientific journals have been issuing new guidance aiming to improve the quality and reproducibility of the data presented in western blots. With the introduction of automated capillary western blotting this goal now seems to be within reach.

In 2015, theWe-Met Functional Biochemistry Facilitywas created within theInstitute of Metabolic and Cardiovascular Diseases (I2MC)in Toulouse with the aim of providing the very latest innovative technologies and technical support around functional biochemistry. Particular focus was given to three major innovative technologies around automated western blotting, homogeneous time resolved fluorescence (HTRF)-mediated biomarker discovery and the study of cell metabolism. The facility now boasts 80 users, including collaborations in academic and private research.

We recently had the pleasure of speaking with Dr Alexandre Lucas, founder and core manager of the We-Met Facility, about the automation of western blotting, theautomated SimpleWestern platforms Wes and Jess from ProteinSimple, a Bio-Techne brand, they utilize at the facility and the difference these advances are making to research.

Karen Steward (KS): How do the automated western blot systems Wes and Jess differ to traditional manual western blotting?

Alexandre Lucas (AL):Classic western blotting is a method that has changed little over the past 30 years, with the exception of the advent of the charge-coupled device (CCD) camera replacing autoradiographic film. The arrival of the automated capillary western is a real technological leap.

In fact, this method differs little in principle as we retain the three major key steps of western blotting: separation, immobilization/transfer and immunorevelation. But since these steps are automated, we gain in reproducibility and the availability of time to the user. In addition, the passage through the capillaries can significantly reduce the amount of sample required.

KS: Can antibodies used in traditional systems be used for the automated systems, or are specialist reagents required?

AL:Yes, without any problem. Of the 200 antibodies we had to develop on the platform over the past 3 years, only 10% of these antibodies could not be transferred.

We work with all existing antibody suppliers without restriction. The flexibility of the device across all reagents allows us to optimize each step very efficiently. There is a big community database available with all the antibodies validated by the users. We don’t need any specialist reagents. This is one of the huge advantages of this technology, everything is open and customizable!

KS: Traditional western blots often require a lot of optimization to produce a good quality blot. Is this the same case for automated western blot systems, such asJess?

AL:As with traditional western blots, if you want a high-quality quantification, you need to optimize. The major thing is you can test many conditions to obtain good linearity and saturation conditions in only 1 run of 25 capillaries in 3 hours.

You can’t test so many different conditions in a traditional western blot and on many occasions, those who use our facility re-learn the meaning of “gold standards” when it comes western blot quantification, with Jess.

KS: How has automated western blotting helped your research? Can you please tell us about any specific areas in which this technology has facilitated work that was not otherwise possible?

AL:杰斯已经完全重新思考th成为可能e western blot within the institute, but also for the various start-ups in the Toulouse region. It has helped us in many projects in cardiology, neuroscience, metabolism and even cosmetology. The reproducibility, the small amount of material required and its flexibility have enabled us to carry out large-scale clinical studies or even to produce a western blot on a protein of 1 kDa to 440 kDa.

Dr Alexandre Lucas was speaking to Dr Karen Steward, Senior Science Writer for Technology Networks.