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.
Technology Networks recently had the pleasure of speaking with Dr Alexandre Lucas, founder and core manager of the We-Met Functional Biochemistry Facility within the Institute of Metabolic and Cardiovascular Diseases (I2MC) in Toulouse, about the automation of western blotting and the difference this advance is making to research.
Karen Steward (KS): How do automated western blot systems differ from 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 charge-coupled device (CCD) camera replacing autoradiographic film. The arrival of the automated western in capillary is a real technological leap.
In fact, this method differs little in principle since 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 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 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 blotting?
AL: In terms of traditional western blots, if you want 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 in many cases the users at our facility re-learn the basics of western blot quantification gold standards with automated western blotting.
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: The automated western has made it possible to completely rethink the 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, Science Writer for Technology Networks.
This content was originally published here.