[Demelerlab] update on AUC experiment

[Borries Demeler]

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