The first French congress on Integrative Structural Biology (BSI) conference was hosted in Tolouse in 2019 by the French Association of Crystallography (AFC) and the French Society of Biophysics (SFB).
The meeting brought together biologists from across Europe to share their latest work.
Fluidic Analytics presented their latest data from the Fluidity One-W instrument for protein interactions analysis, followed by a Q&A session with Jonathan Faherty - applications specialist.
Question: How does the Fluidity One-W detect the proteins in the diffusion chambers?
Answer: Only the protein of interest is labelled with a Fluorophore, so only one of your two interacting proteins is labelled. As a result, the labelled protein is the only protein you detect in that particular solution. If you have an Alexa Fluor 488 dye on your protein, for example, that will be detected by the system in those two diffusion chambers.
Question: How well are you able to deal with mixtures? For example, if you have a number of components in solution, how well can you measure the properties of each of them?
Answer: That depends on the labelling, if you have one particular protein label then the other proteins in the mixture are not relevant. If a protein has a different number of oligomeric states and as a result are all labelled, then you will end up getting a wider range for the average hydrodynamic radius.
Question: Does the Fluidity One-W have a limitation in terms of protein concentration?
Answer: The lower limit is around sub one hundred picomolar and the upper limit allows for the protein to be in micromolar. In some cases, it may also be possible to treat the label as a tracer for a larger protein. If you do that then there's a possibility to go up to millimolar.
Question: Do you have experience using proteins that have an elongated shape? So that the hydrodynamic radius is not relative to the mass.
Answer: In those cases, it depends on how well you know your protein. If you know your protein is non-globular and it has a more elongated shape, then you can account for that and expect the results to have a slightly larger hydrodynamic radius range. In terms of interpreting size into mass, the system gives you a range, for example 1 kDa to 20 kDa and then you take your own knowledge and apply it to the results to see if the range is accurate.
Question: You said the auxiliary stream in the chip is made of water, what if the protein of interest is not happy in water.
Answer: One thing to bear in mind is that the time proteins spend diffusing across the auxiliary fluid is extremely short. Proteins spend approximately one second in that diffusion channel, so even if the protein starts to denature it won't matter. You are looking for the detection of the protein in the diffusion chamber. So even if it has denatured it doesn't matter since it's already diffused and is then detected.
If you have a question of your own, get in touch to ask our scientific team here.