[Demelerlab] [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

[Martin, Reece]

Hi Vince and Andres,

Thank you for your responses. I have everything that I need to run the first experiment. I will update once it is completed, and we can go from there.

Vince, in regard to the higher temperature studies with CanA, the maximum temperature the instrument is capable of running at is 40 degrees Celsius. So, we could run the instrument at 37 degrees Celsius with the sample. However, if we were expecting to see oligomerization as a result of heating to (or being held at) this temperature, then we would need to first incubate with calcium ions at 37 degrees Celsius. This could be achieved transiently through a heating block for an hour before sample loading, or more slowly via the AUC as it heats to 37 degrees Celsius over the course of roughly one hour (Bo, please correct me if this is wrong). The AUC then has a temperature-equilibration delay time which we can set. The timescale of the oligomerization would also need to be considered for the temperature-equilibration delay as significant oligomerization during the course of the AUC experiment would yield uninterpretable results. However, if it is the act of cooling to ambient temperature that induces oligomerization, then we would incubate at 37 degrees Celsius with calcium ions for an hour, allow the sample to cool to ambient temperature, and then run at ambient temperature. Perhaps you or Bo have better insight into which of these methods would be more suitable. If not, I can certainly try both.


Cheers,

Reece
________________________________
From: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>
Sent: 07 September 2023 12:58 PM
To: Borries Demeler <demeler at gmail.com>; Conticello, Vincent <vcontic at emory.edu>
Cc: Martin, Reece <reece.martin at uleth.ca>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins


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Hi everyone,

I just wanted to add the last details. I included some tubes containing the exact buffer in which the proteins are found and another tube with the buffer with no EDTA. In short, the proteins are in a low salt buffer solution (80mM NaCl, 50mM Tris/HCl pH= 7.5, 9% glycerol and 0.01 mM EDTA) thus they all contain 0.01mM EDTA.

Let me know if anything else is needed.
Best,
Andres
________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Thursday, September 7, 2023 2:44 PM
To: Conticello, Vincent <vcontic at emory.edu>
Cc: Martin, Reece <reece.martin at uleth.ca>; Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>
Subject: Re: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

This sounds good to me. Glad to hear the proteins were not damaged by the elevated temperature storage conditions. We will commence our studies using the samples you provided. The last thing we need to know is what the EDTA concentrations in the buffer are of the samples you provided to us.

-Borries

On Thu, Sep 7, 2023 at 10:55 AM Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>> wrote:
Hi Reece. The samples should be good since the proteins are thermostable, that is, from a hyperthermophilic organism even though expressed in E. coli. Andres can answer the first part of your question regarding the buffers since he prepped the samples. For the initial experiments in the presence of calcium, the samples should be incubated at ambient temperature in the presence of calcium ion, probably for an hour. CanA might not oligomerize under these conditions, while the Hyper2 sample should. If the initial calcium experiments do not demonstrate oligomerization, the Hyper2 sample can be incubated for 24 hours at ambient temperature. I would not heat the Hyper2 sample since it might rapidly polymerize (based on our experience). With regard to the CanA sample, I would suggest using the 10 mM or maybe 20 mM calcium ion concentration at higher temperature. If we wanted to detect the presence of oligomers, we might not want to heat the sample to higher temperatures (typically we use 80C for filament formation followed by cooling to ambient to induce polymerization).I think that 37 degrees is a good compromise. Would you run the instrument with the sample at 37 or incubate at 37 and run the sample at ambient? If the latter, it may be best to start out at 1 hr incubation at elevated temp. Best, Vince

Vincent P. Conticello, Ph.D.
Professor
Department of Chemistry
Emory University
________________________________
From: Martin, Reece <reece.martin at uleth.ca<mailto:reece.martin at uleth.ca>>
Sent: Wednesday, September 6, 2023 7:11 PM
To: Gonzalez Socorro, Andres <andres.gonzalez.socorro at emory.edu<mailto:andres.gonzalez.socorro at emory.edu>>; Conticello, Vincent <vcontic at emory.edu<mailto:vcontic at emory.edu>>
Cc: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>
Subject: [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins

Hi Vince and Andres,

Considering that the samples arrived at room temperature (the exact length of time is unknown), and that an ice pack leaked (the samples appear to have been sealed properly). We are just looking to confirm that you feel it is worth continuing to process this batch of samples, taking into account your insight into the temperature-stability of the proteins at ambient temperature.

If so, as per the updated experimental design in the LIMS, we will start out running both CanA and Hyper2 at a high and low concentration in the EDTA-containing buffer at 20 degrees Celsius. To accomplish this, I need to know what buffer each of the protein stocks are in and whether or not they contain EDTA.

If this experiment is successful, we will repeat in the presence of calcium ions (10mM for CanA, and 1mM for Hyper2). For this we need to know at what temperature and for how long the samples should be incubated with the CaCl2 solution. Finally, if this experiment is successful, we will look at repeating with increased calcium ion concentrations, as well as running at a higher temperature such as 37 degrees Celsius.

I'll report back on the results from the first run, which will take place this week.

Cheers,

Reece

--
Reece Martin, B.Sc
M.Sc. Candidate
Northwest Biophysics Consortium
Alberta RNA Research and Training Institute (ARRTI)
Lab of Dr. Borries Demeler
Department of Chemistry and Biochemistry
University of Lethbridge
Email: reece.martin at uleth.ca<http://uleth.ca/>
Phone: 403-498-5882
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