In this on-demand webinar, learn more about my experience utilizing methodologies such as MST to monitor and characterize the key factors involved in ribosome assembly. I share examples detailing the fate, binding interactions and structure of immature particles that accumulate in assembly factor knockout or depletion strains.
Initially, a series of binding assays were performed by incubating ribosome samples and potential binding partners, separating the bound from the unbound species, and then utilizing gel electrophoresis to analyze the various protein-ribosome interactions. These assays consumed large amounts of sample, were tedious, time consuming and ultimately led to results that were difficult to interpret. At the end of the day (or years!), we had a lot of interesting data, but did not quite know what to make of it. Fortunately, a top bioanalytics professor at the University of Toronto had recently acquired the Monolith, which enabled researchers to use MST for assessing binding interactions. Binding analysis on the Monolith required extremely low amounts of sample, was performed completely in solution, was fast and easy to use. These were all prominent features that were important for driving our research forward and therefore we were excited to jump into binding affinity experiments on the Monolith.
Establishing if two molecules interact with each other and characterizing how “tight” that interaction is can be a very challenging task. Often researchers can spend months or years preparing their samples and running rather crude binding assays to assess the various binding interactions of interest. An example of this binding affinity characterization assay is when two potential binding partners are incubated together to allow them bind to each other, and then run through a gel by electrophoresis to assess the interaction. There are several problems with this type of experimental set up, including non-equilibrium conditions, limitations in the concentrations of sample that can be used and difficulty quantifying the binding interaction. For these reasons, analyzing protein-RNA interactions involved in ribosome assembly can be a daunting task. To circumvent these problems, we turned to a technique known as MicroScale Thermophoresis (MST), which enabled rapid characterization of challenging binding interactions involved in the biogenesis of the ribosome.
