[Demelerlab] update on AUC experiment

[Borries Demeler]

Justin,
I set up a zoom call for Tuesday at 1 pm and sent out an invite. Can you
please share this invite with your student and ask him to join as well?
Thanks! -Borries

On Fri, Jul 7, 2023 at 7:56 AM Pahara, Justin (AAFC/AAC) <
justin.pahara at agr.gc.ca> wrote:

> 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> 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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