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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</div>
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<div class="PlainText">Vincent P. Conticello, Ph.D.<br>
Professor<br>
Department of Chemistry<br>
Emory University</div>
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<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> Martin, Reece <reece.martin@uleth.ca><br>
<b>Sent:</b> Wednesday, September 6, 2023 7:11 PM<br>
<b>To:</b> Gonzalez Socorro, Andres <andres.gonzalez.socorro@emory.edu>; Conticello, Vincent <vcontic@emory.edu><br>
<b>Cc:</b> Borries Demeler <demeler@gmail.com>; demelerlab@biophysics.uleth.ca <demelerlab@biophysics.uleth.ca><br>
<b>Subject:</b> [External] Analytical Ultracentrifugation Analysis of Cannula-mimetic Proteins</font>
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Hi Vince and Andres,</div>
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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. <span class="x_ContentPasted2" style="display:inline!important; color:rgb(0,0,0); background-color:rgb(255,255,255)"></span></div>
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If so, as per the updated experimental design in the LIMS, we will start out running both<span style="display:inline!important; color:rgb(0,0,0); background-color:rgb(255,255,255)"><span> </span>CanA and Hyper2</span> at a high and low concentration in the
EDTA-containing buffer at 20 degrees Celsius. To accomplish this, I need to know w<span class="x_ContentPasted3" style="display:inline!important; color:rgb(0,0,0); background-color:rgb(255,255,255)">hat buffer each of the protein stocks are in and whether
or not they contain EDTA. </span> </div>
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<span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:12pt">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.</span><br>
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<span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:12pt">I'll report back on the results from the first run, which will take place this week.</span></div>
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