In an expert webinar now available on demand, Dr. Sean Devenish reviews the latest results generated using the new technique of microfluidic diffusional sizing (MDS) across protein analysis, from QC to aggregation and interactions. Additionally, he discusses future developments that MDS will bring to the field and how these could impact your research.
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Q: Can I use MDS to monitor DNA or RNA binding to a protein?
SD: Absolutely. There's a couple of different ways of looking at this – if you're really interested in the DNA or RNA binding to the protein, then that would point towards the Fluidity One-W using fluorescently labelled nucleic acid oligomers, as in the aptamer example I presented during the webinar.
If your more interested in protein binding to DNA such as looking at transcription factors binding to DNA and it's maybe a bit semantic, but I would say there's a bit of a difference, then you could use the Fluidity One with unlabeled protein and nucleic acid.
DNA is essentially invisible on the Fluidity One as it isn’t efficiently labelled by the on-board dye, so on that instrument you could look selectively at the protein. You'd have a constant concentration of protein and titrate in your DNA, and you would see a size change which would reflect the interaction between the protein and the DNA. You could also look at the change in size to confirm that the protein is binding to a single piece of DNA. So, absolutely.
Q: What about highly hydrophobic proteins?
SD: We have a surface treatment on our chips which means that even proteins that are typically considered to be sticky aren’t a problem when measuring on our instrument. I should mention that some of the peptides we were measuring (tryptophan- and tyrosine-free peptides) were reported to be terribly sticky, but we got good results, irrespective of the fact that they were difficult to work with.
Q: What is the diameter range one can measure virus preparations?
SD: The usable diameter range for the instruments is from 0.5 nanometers up to 20 nanometers; or in real terms, from di- or tripeptides at the lowest end, right up to the E. coli ribosome. For viruses, it would depend on which virus you are interested in. Some of them might fit within the range, but some of them certainly are very large and would not be something that we could size with our current technology.
Q: My samples often contain ethanol as an additive. Will this affect the results?
SD: Ethanol is tolerated and up to 5% with no effect on the results. Likewise, DMSO and even glycerol can be tolerated in samples. We are in the process of putting together a more comprehensive list of additives that can be incorporated in samples.
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