[Demelerlab] update on AUC experiment

[Pahara, Justin (AAFC/AAC)]

Good Morning Borries,

Good question – we had not planned any purification at this point. The reason being we are ultimately interested in aptamers that bind to different types of macromolecules within the lysate (protein, carbs, etc). If we run a purification it will bias toward a particular system. I was hoping we could do a one pot process during this prototype experiment.

It might be possible to label the aptamer with both biotin and a fluorophore so that we can pull out the aptamers with beads… what do you think? I was hoping to start with a simple experiment and add complexity (and expense) as needed, so what do you think is the simplest starting point??

A call next week sounds great – how about Tuesday? 1pm?


Best wishes,
Justin.

From: Borries Demeler <demeler at gmail.com>
Sent: Tuesday, July 4, 2023 3:02 PM
To: Pahara, Justin (AAFC/AAC) <justin.pahara at AGR.GC.CA>; demelerlab at biophysics.uleth.ca
Subject: Re: update on AUC experiment

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Hi Justin,
this sounds like a great start! So many opportunities for applying multi-wavelength AUC. I was wondering what level of purification, if any, has been done on these complexes. Potentially a sucrose gradient could be used to concentrate some of the complexes. It would be wonderful if we could get the labeled molecules by themselves as controls, both to determine their sedimentation coefficients and their absorbance spectra when they are pure so we can distinguish them from the rest of the proteins in your lysate, are there any purifications planned?

Perhaps we could set up a Zoom call next week with the rest of the lab members and your ULeth student?

-Borries

On Tue, Jul 4, 2023 at 1:11 PM Pahara, Justin (AAFC/AAC) <justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>> wrote:
Good Afternoon Borries,

I hope you’re enjoying a good start to summer 😊

I want to provide a quick update about the research we discussed earlier. We have begun to prototype our first SELEX experiment where we’ve designed a ssDNA aptamer library and we plan to run a mock experiment with whole cell lysate from E. coli expressing RFP or CFP and then try absorbance/fluorescence ultracentrifugation with you to identify fraction(s) of Aptamer-RFP. We could also use chromoproteins if that is better and you’d prefer AUC. If possible it would be great to not have the aptamer fluorescently tagged for this first experiment.

Our initial aptamer library is 90 nts long with 50 Ns, so ~30 kDa and the RFP/CFP is about the same, so we’re looking at around ~60 kDA for single DNA/protein interaction.

What do you think? Might we be able to try it out later this month? The summer student who is working on the project is a ULeth student and so he could do most of the work if your team is busy!

Best,
Justin.


Dr. Justin Pahara

Research Scientist and Project Lead

Nanotechnology (Biotic Stresses and Adaptation)
Agriculture and Agri-Food Canada / Government of Canada
justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>

Nanotechnologie (Adaptation et Contraintes Biotiques)
Agriculture et Agroalimentaire Canada / Gouvernement du Canada
justin.pahara at agr.gc.ca<mailto:justin.pahara at agr.gc.ca>
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