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Hi Borries
<div>For standard NMR tubes we typically make up 600-650 uL of the solution.</div>
<div>Then we put 550uL in the NMR tube</div>
<div>But this work is being done in a Shegimi tube, which uses far less volume, as it restricts the sample to the active coil area only. This increases the sensitivity and the glass in the shegimi tube is also susceptibility matched for d2o/h2o which allows
for better shimming.</div>
<div>For these tubes we need around 280-320uL of sample. Maybe plan for 350uL just to be safe.</div>
<div>Mike, can you please confirm if I am correct on the shegimi tube volumes ?</div>
<div>Cheers</div>
<div>Tony <br>
<br>
<div dir="ltr">
<div>Tony Montina</div>
<div>Director, Science Operations</div>
<div>Director, Magnetic Resonance Facility</div>
<div>Faculty of Arts and Science</div>
<div>Instructor, <span style="font-size: 13pt;">Department of Chemistry and Biochemistry</span></div>
<div>University of Lethbridge</div>
<div>SA6214, Science Commons </div>
<div>4401 University Drive West</div>
<div>Lethbridge, Alberta, Canada, T1K3M4</div>
<div>Office: 1-403-394-3927</div>
<div>Lab: 1-403-329-2230</div>
<div><br>
</div>
This message has been sent from my mobile device so please ignore any autocorrect or typing errors.</div>
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<blockquote type="cite">On Mar 21, 2021, at 4:08 PM, Borries Demeler <demeler@gmail.com> wrote:<br>
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</div>
<blockquote type="cite">
<div dir="ltr"><span>Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing@uleth.ca.</span><br>
<span></span><br>
<span></span><br>
<span>Thank you, Tony!</span><br>
<span>Can you remind me of the volume needed to do the experiment you planned?</span><br>
<span>This will determine if we need Bruno to send more protein.</span><br>
<span></span><br>
<span>Thanks, -Borries</span><br>
<span></span><br>
<span>On Sun, Mar 21, 2021 at 06:43:18PM +0000, Montina, Tony wrote:</span><br>
<blockquote type="cite"><span>Hello All,</span><br>
</blockquote>
<blockquote type="cite"><span>I agree with both Paul and Borries.</span><br>
</blockquote>
<blockquote type="cite"><span>The higher we can get the myotoxin concentration in SDS the better for signal to noise and overall NMR experiment time in our room temperature probe. Also, as Paul and Borries both mentioned, using a 5mM concentration of the buffer
and also a significantly lower SDS concentration (at least one order of magnitude but potentially two) will greatly improve the dynamic range of the ADC convertor and hence should result in better signal to noise (and possibly resolution).</span><br>
</blockquote>
<blockquote type="cite"><span>The last experiments we used ~2.5mg/mL concentration and our first experiments used 4 mg/ml.</span><br>
</blockquote>
<blockquote type="cite"><span>This was because things started precipitating out at any higher of concentration. Hopefully this was due to the micelle formation and once this problem is gone - now that we are working with SDS below the CMC - we can get myotoxin
into solution at much higher concentrations.</span><br>
</blockquote>
<blockquote type="cite"><span>First, Borries and Amy can determine the minimum amount of SDS required that is below the 0.2% CMC of SDS.</span><br>
</blockquote>
<blockquote type="cite"><span>I would then suggest that Amy and Mike use this SDS concentration to determine the max solubility of the myotoxin in that SDS concentration and 5mM buffer (but not exceed 20mg/mL of myotoxin as suggested by Borries).</span><br>
</blockquote>
<blockquote type="cite"><span>Once this is done, we can re-run the NMR experiments on the SDS (at lower concentration) + Myotoxin (hopefully at a higher concentration) in 5mM buffer. Hopefully we can get the concentration of myotoxin up much higher than 4mg/mL
so that we can run these experiments quickly.</span><br>
</blockquote>
<blockquote type="cite"><span>Amy, do we have enough myotoxin on hand in Lethbridge to go up to 20mg/mL?</span><br>
</blockquote>
<blockquote type="cite"><span>Lastly, I agree that a NOESY experiment will be useful, but that would be more useful for the later paper and depending on the final concentration of myotoxin we are able to use in SDS, may or may not be possible.</span><br>
</blockquote>
<blockquote type="cite"><span>For example, if this is a 2 week long NOESY experiment we may not be able to run this on our room temperature probe. Increasing the concentration will help greatly with this problem.</span><br>
</blockquote>
<blockquote type="cite"><span>I hope that helps clear up the sample prep questions for NMR.</span><br>
</blockquote>
<blockquote type="cite"><span>Amy, please reach out to Mike and I when you are ready next week and we can get the new sample prepared.</span><br>
</blockquote>
<blockquote type="cite"><span>This is really good timing, as I believe Mike has booked the NMR instrument for the entirety of next weekend.</span><br>
</blockquote>
<blockquote type="cite"><span>Cheers</span><br>
</blockquote>
<blockquote type="cite"><span>Tony</span><br>
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<blockquote type="cite"><span>________________________________________</span><br>
</blockquote>
<blockquote type="cite"><span>Tony Montina</span><br>
</blockquote>
<blockquote type="cite"><span>Director | Science Operations</span><br>
</blockquote>
<blockquote type="cite"><span>Director | Magnetic Resonance Facility</span><br>
</blockquote>
<blockquote type="cite"><span>Instructor | Department of Chemistry and Biochemistry</span><br>
</blockquote>
<blockquote type="cite"><span>Faculty of Arts and Science</span><br>
</blockquote>
<blockquote type="cite"><span>The University of Lethbridge</span><br>
</blockquote>
<blockquote type="cite"><span>Lethbridge, Alberta, Canada, T1K 3M4</span><br>
</blockquote>
<blockquote type="cite"><span>Office: 1-403-394-3927</span><br>
</blockquote>
<blockquote type="cite"><span>Lab: 1-403-329-2230</span><br>
</blockquote>
<blockquote type="cite"><span></span><br>
</blockquote>
<blockquote type="cite"><span>-----Original Message-----</span><br>
</blockquote>
<blockquote type="cite"><span>From: Myotox <myotox-bounces@biophysics.uleth.ca> On Behalf Of Hazendonk, Paul</span><br>
</blockquote>
<blockquote type="cite"><span>Sent: March 20, 2021 10:41 PM</span><br>
</blockquote>
<blockquote type="cite"><span>To: Myotoxin-II discussion <myotox@biophysics.uleth.ca></span><br>
</blockquote>
<blockquote type="cite"><span>Subject: Re: [Myotox] myotoxin2-SDS titration</span><br>
</blockquote>
<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>As high a conc of protein as is possible, considering aggregation issues etc, etc. It will really speed things up especially the 2D experiments.</span><br>
</blockquote>
<blockquote type="cite"><span></span><br>
</blockquote>
<blockquote type="cite"><span>-----Original Message-----</span><br>
</blockquote>
<blockquote type="cite"><span>From: Myotox <myotox-bounces@biophysics.uleth.ca> On Behalf Of Borries Demeler</span><br>
</blockquote>
<blockquote type="cite"><span>Sent: March 20, 2021 9:38 PM</span><br>
</blockquote>
<blockquote type="cite"><span>To: Myotoxin-II discussion <myotox@biophysics.uleth.ca></span><br>
</blockquote>
<blockquote type="cite"><span>Subject: Re: [Myotox] myotoxin2-SDS titration</span><br>
</blockquote>
<blockquote type="cite"><span></span><br>
</blockquote>
<blockquote type="cite"><span>Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing@uleth.ca.</span><br>
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<blockquote type="cite"><span>If this is possible that would be very exciting! Paul, can you please indicate the concentration and volume of protein needed to perform these experiments. We will then test by AUC how low we can go with SDS and still see the shift
in s.</span><br>
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<blockquote type="cite"><span>-Borries</span><br>
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<blockquote type="cite"><span>On Sun, Mar 21, 2021 at 02:36:30AM +0000, Paul Hazendonk wrote:</span><br>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>This is very exciting. May I suggest we also try a NOESY experiment one we have done the analysis on the low sds conc. Sample since if the binding is very specific we may be able to identify the amino acids with which it is interacting.</span><br>
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<blockquote type="cite"><span>From: Myotox <myotox-bounces@biophysics.uleth.ca> On Behalf Of Bruno</span><br>
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</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Lomonte</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>Sent: March 20, 2021 4:40 PM</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>To: myotox@biophysics.uleth.ca</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>Subject: Re: [Myotox] myotoxin2-SDS titration</span><br>
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<blockquote type="cite"><span></span><br>
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</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Caution: This email was sent from someone outside of the University of Lethbridge. Do not click on links or open attachments unless you know they are safe. Suspicious emails should be forwarded to phishing@uleth.ca<mailto:phishing@uleth.ca>.</span><br>
</blockquote>
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<blockquote type="cite"><span>Interesting as always, Borries and all ! the combination of your biophysical analyses + NMR are providing lots of exciting new information, as you explained. I will send more protein this week to Amy's address. Thanks a lot for
all the interest and hard work.</span><br>
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<blockquote type="cite"><span>Best regards,</span><br>
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<blockquote type="cite"><span>Bruno</span><br>
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<blockquote type="cite"><span>+++++++</span><br>
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<blockquote type="cite"><span>On 3/20/2021 11:20 AM, Borries Demeler wrote:</span><br>
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<blockquote type="cite"><span>As promised, here is an update on the SDS titration experiment.</span><br>
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<blockquote type="cite"><span>Amy found plenty of Myotoxin for doing these experiments in our</span><br>
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<blockquote type="cite"><span>freezer and performed an AUC experiment doing a titration with very</span><br>
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<blockquote type="cite"><span>low</span><br>
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<blockquote type="cite"><span>concentration of SDS (0.01 and 0.02 percent of SDS, which should be</span><br>
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<blockquote type="cite"><span>well</span><br>
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<blockquote type="cite"><span>below the CMC of SDS). This clearly shows that there is a significant</span><br>
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<blockquote type="cite"><span>change in the sedimentation profile (see attached).</span><br>
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<blockquote type="cite"><span>A few notes:</span><br>
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<blockquote type="cite"><span>1. The monomer control was actually run at 300 nm with ~21 mg/ml</span><br>
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<blockquote type="cite"><span>concentration - so very high, and no evidence of dimerization there.</span><br>
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<blockquote type="cite"><span>The protein concentration in the SDS titration experiments was much,</span><br>
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<blockquote type="cite"><span>much lower at only about 10 uM.</span><br>
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<blockquote type="cite"><span>2. even the 0.01% SDS sample exhibits significant shift in the</span><br>
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<blockquote type="cite"><span>s-values</span><br>
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<blockquote type="cite"><span>which can only be explained by dimerization (as seen in the SDS-PAGE),</span><br>
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<blockquote type="cite"><span>not by micelle formation with multiple myotoxin molecules being</span><br>
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<blockquote type="cite"><span>incorporated into the micelle.</span><br>
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<blockquote type="cite"><span>3. The 0.02% SDS concentration sample sedimented *slower* than the</span><br>
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<blockquote type="cite"><span>0.01% SDS sample. If I think about this it makes sense if we</span><br>
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<blockquote type="cite"><span>assume that instead of additional oligomerization we simply bind</span><br>
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<blockquote type="cite"><span>more SDS to the complex, because SDS could significantly affect</span><br>
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<blockquote type="cite"><span>friction. It also affects the partial specific volume. It is less</span><br>
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<blockquote type="cite"><span>dense than protein so the overall partial specific volume of the</span><br>
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<blockquote type="cite"><span>complex becomes less dense as more SDS is bound.</span><br>
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<blockquote type="cite"><span>4. There isn't just one peak visible, consistent with either monomer</span><br>
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<blockquote type="cite"><span>or</span><br>
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<blockquote type="cite"><span>dimer, but there are at least 2 major peaks in both samples,</span><br>
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<blockquote type="cite"><span>suggesting</span><br>
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<blockquote type="cite"><span>possible even high order structures than dimer are present, or at</span><br>
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<blockquote type="cite"><span>least</span><br>
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<blockquote type="cite"><span>some equilibrium between at least two oligomeric structures. It is not</span><br>
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<blockquote type="cite"><span>clear to me what oligomers are present, but with a little additional</span><br>
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<blockquote type="cite"><span>AUC work we could probably figure this out.</span><br>
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<blockquote type="cite"><span>I think this brings up more questions, but also answers one important</span><br>
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<blockquote type="cite"><span>question for Paul and Tony:</span><br>
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<blockquote type="cite"><span>We can definitely significantly reduce the SDS concentration to avoid</span><br>
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<blockquote type="cite"><span>the strong overlay signals from SDS in the NMR experiments. Also, we</span><br>
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<blockquote type="cite"><span>can keep the very low phosphate concentration (5mM).</span><br>
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<blockquote type="cite"><span>I think before we go to the next NMR experiments I want to do one</span><br>
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<blockquote type="cite"><span>more AUC experiment: Amy, can you please find out the lowest possible</span><br>
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<blockquote type="cite"><span>SDS concentration that causes a shift for a given protein concentration?</span><br>
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<blockquote type="cite"><span>What I found surprising is that the 0.02% solution sedimented slower</span><br>
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<blockquote type="cite"><span>than the 0.01%, this suggests the formation of discrete species, with</span><br>
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<blockquote type="cite"><span>tight SDS binding, and no change in oligomerization between the two</span><br>
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<blockquote type="cite"><span>SDS</span><br>
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<blockquote type="cite"><span>concentrations. That's important. I would like to know if we can find</span><br>
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<blockquote type="cite"><span>out the lowest SDS concentration that causes this behavior. I think</span><br>
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<blockquote type="cite"><span>the SDS binds very tightly, so do we see the same behavior at 0.001%</span><br>
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<blockquote type="cite"><span>SDS? I think we should do a titration one order of magnitude lower,</span><br>
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<blockquote type="cite"><span>and at the same time increase the protein concentration. THis would</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>tell us two things: 1. what is the best ratio of high concentration</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>protein and low concentration SDS that clearly starts showing this</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>oligomerization? That ratio would be best for NMR, maximum protein</span><br>
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<blockquote type="cite"><span>signal,</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>minimum SDS signal. Let's find the critical ratios with AUC. We may</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>have</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>to run the SDS titration with more points and also repeat it at</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>multiple protein concentrations. I would also like to know the maximum</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>s-value shift that can be observed, and whether the 2-peak pattern</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>persists.</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>Paul/Tony: What protein concentration would you like to run ideally?</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>1 mg/ml, 10 mg/ml, 20 mg/ml? Since Bruno is willing to send more</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>sample,</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>I think we can play with this ratio a bit. Amy, how much is left?</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite"><span>For AUC I would prefer not to go much above 20 mg/ml to avoid</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>concentration dependent non-ideality effects.</span><br>
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<blockquote type="cite"><span>Let me know what y'all think about this approach.</span><br>
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<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
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</blockquote>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>-Borries</span><br>
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<blockquote type="cite"><span></span><br>
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</blockquote>
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<blockquote type="cite"><span></span><br>
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</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>_______________________________________________</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Myotox mailing list</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Myotox@biophysics.uleth.ca<mailto:Myotox@biophysics.uleth.ca></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>--</span><br>
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</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
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<blockquote type="cite">
<blockquote type="cite"><span>Bruno Lomonte, Ph.D.</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Instituto Clodomiro Picado</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
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<blockquote type="cite">
<blockquote type="cite"><span>Facultad de Microbiología</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
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<blockquote type="cite">
<blockquote type="cite"><span>Universidad de Costa Rica</span><br>
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</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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</blockquote>
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<blockquote type="cite"><span>San José, COSTA RICA</span><br>
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<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
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<blockquote type="cite">
<blockquote type="cite"><span>tel.of. (+506) 2511 7888</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span></span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>bruno.lomonte@ucr.ac.cr<mailto:bruno.lomonte@ucr.ac.cr></span><br>
</blockquote>
</blockquote>
<blockquote type="cite"><span></span><br>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>_______________________________________________</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Myotox mailing list</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>Myotox@biophysics.uleth.ca</span><br>
</blockquote>
</blockquote>
<blockquote type="cite">
<blockquote type="cite"><span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
</blockquote>
</blockquote>
<blockquote type="cite"><span></span><br>
</blockquote>
<blockquote type="cite"><span>_______________________________________________</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox mailing list</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox@biophysics.uleth.ca</span><br>
</blockquote>
<blockquote type="cite"><span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
</blockquote>
<blockquote type="cite"><span>_______________________________________________</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox mailing list</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox@biophysics.uleth.ca</span><br>
</blockquote>
<blockquote type="cite"><span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
</blockquote>
<blockquote type="cite"><span>_______________________________________________</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox mailing list</span><br>
</blockquote>
<blockquote type="cite"><span>Myotox@biophysics.uleth.ca</span><br>
</blockquote>
<blockquote type="cite"><span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
</blockquote>
<span>_______________________________________________</span><br>
<span>Myotox mailing list</span><br>
<span>Myotox@biophysics.uleth.ca</span><br>
<span>https://demeler7.uleth.ca/mailman/listinfo/myotox</span><br>
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