[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Tue Jul 25 17:18:27 MDT 2023

Here is the graph with the zoomed in secton. I did also notice in the Excel sheet that after about 280-285 nm the values start to get negative so I think it will be best to remeasure these.

[cid:c9480f21-e6a0-4b77-a600-90c0e8d2f058]
________________________________
From: Demelerlab <demelerlab-bounces at biophysics.uleth.ca> on behalf of Henrickson, Amy <amy.henrickson at uleth.ca>
Sent: Tuesday, July 25, 2023 5:06 PM
To: Borries Demeler <demeler at gmail.com>
Cc: James S. Nowick <jsnowick at uci.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>; Jason Zhu <jasonz13 at uci.edu>
Subject: Re: [Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

We do have a cuvette that only needs 60uL of samples. It does have a little more noise than the ones we usually use but for getting in idea of OD it should work fine.

-Amy

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________________________________
From: Borries Demeler <demeler at gmail.com>
Sent: Tuesday, July 25, 2023 4:59:24 PM
To: Henrickson, Amy <amy.henrickson at uleth.ca>
Cc: James S. Nowick <jsnowick at uci.edu>; Jason Zhu <jasonz13 at uci.edu>; demelerlab at biophysics.uleth.ca <demelerlab at biophysics.uleth.ca>; Ranasinghe, Maduni <maduni.ranasinghe at uleth.ca>
Subject: Re: AUC Collaboration: Sizing Amyloid Beta derived Oligomers

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Amy, keep in mind that some of the samples at ~110 ul for the 3 mm centerpieces may be too low volume for our 1 cm pathlength cuvettes, and I don't think we want to dilute until we have enough volume so we can still measure at high enough concentration. As James suggested, interference could be an option, though I am not sure if the 3 mm centerpieces actually are in the correct focus plane for the interference optics, something we need to confirm first. They should be in the 2/3 plane, but are in the 0.5 plane. Maybe this discrepancy is not critical, and there is enough material so we can make this measurement. Either way, we'll figure it out, just something to think about.

Jason, assuming the Y-axis is correct, can you please send us a plot where the Y-axis is zoomed to 0-3 OD region because anything over 3 OD in a 10 mm centerpiece would be too absorbing to be measured in a 3 mm centerpiece.

Thanks! -Borries

On Tue, Jul 25, 2023 at 4:41 PM Henrickson, Amy <amy.henrickson at uleth.ca<mailto:amy.henrickson at uleth.ca>> wrote:
Hello,

I think that we will just collect spectra of the samples on our instrument before we measure them anyway. This will likely be easier for everyone.
Jason sent a document saying how much water to resuspend every sample in so we can do that and measure them to see what wavelength we should measure at.

Regarding the dissolving of the sample, I know that it said the type of water was important. We have milli-q water we could use of water for injection. Which would be better?

Thanks,

Amy HEnrickson
________________________________
From: James S. Nowick <jsnowick at uci.edu<mailto:jsnowick at uci.edu>>
Sent: Tuesday, July 25, 2023 3:20 PM
To: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Cc: Jason Zhu <jasonz13 at uci.edu<mailto:jasonz13 at uci.edu>>; Henrickson, Amy <amy.henrickson at uleth.ca<mailto:amy.henrickson at uleth.ca>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca> <demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>>; Ranasinghe, Maduni <maduni.ranasinghe at uleth.ca<mailto:maduni.ranasinghe at uleth.ca>>
Subject: Re: AUC Collaboration: Sizing Amyloid Beta derived Oligomers

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Hi Borries,

I’m pretty sure these a run on the NanoDrop spectrophotometer. The NanoDrop has a variable pathlength of 0.03-1.0 mm and typically normalizes the Y-axis to the equivalent of a 1.0 cm pathlength. As a result, it can give readings for concentrated solutions of up to 550 absorbance units. .

Best,
James

On Jul 25, 2023, at 1:55 PM, Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>> wrote:

I am wholly confused about the y axis scale and label on your plot. It says 10 mm absorbance (i.e., 1 cm cuvette?). You should get values in the 0.0-1.0 range, not in the hundreds of OD. Assuming all of these measurements are within the dynamic range of the detector, we should be able to use any wavelength between 210-240 nm. We will adjust the wavelength so that any concentration will absorb within 0.3-0.7 OD in a 3 mm centerpiece. Good to know that water+TFA against water does not absorb at all, thanks for measuring this. It would still help us to know what the actual scale on the y axis is. Can you send a corrected plot?

Thanks! -Borries

On Tue, Jul 25, 2023 at 1:36 PM Jason Zhu <jasonz13 at uci.edu<mailto:jasonz13 at uci.edu>> wrote:
I reran the UV-vis spectra of water + 0.1% TFA, 1 mM F19Cha, 3 mM F19Cha, and 9 mM F19Cha all blanked against water and it worked much better this time. The TFA absorbs negligibly above ~220 nm and absorbs weakly compared to that of F19Cha. To get a 0.6 absorbance in a 3 mm centerpiece for the 1, 3, and 9 mM samples with less than 0.3 background absorbance, I would recommend 268, 278, and 279 nm respectively. I'm open to adjusting the wavelengths as you see fit. I've attached the Excel for the spectra below. I think the reason why I got negative absorbance last time was because the nanopure water that I was blanking with had some quality control issues.

Please feel free to reach out if there's anything you need from my end!

Best,
Jason
<image.png>

Best,
Jason

On Fri, Jul 21, 2023 at 10:47 AM James S. Nowick <jsnowick at uci.edu<mailto:jsnowick at uci.edu>> wrote:
Wonderful! Thanks, Bruce!
James

On Jul 21, 2023, at 10:21 AM, Bowler, Bruce <Bruce.Bowler at mso.umt.edu<mailto:Bruce.Bowler at mso.umt.edu>> wrote:

Hi Borries,

The samples arrived about an hour ago and are in my -20 freezer.

I will mostly be around my lab until about 2:45 pm today.

Cheers,
Bruce

From: Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>>
Sent: Wednesday, July 19, 2023 9:26 PM
To: Jason Zhu <jasonz13 at uci.edu<mailto:jasonz13 at uci.edu>>
Cc: James S. Nowick <jsnowick at uci.edu<mailto:jsnowick at uci.edu>>; Henrickson, Amy <amy.henrickson at uleth.ca<mailto:amy.henrickson at uleth.ca>>; demelerlab at biophysics.uleth.ca<mailto:demelerlab at biophysics.uleth.ca>; maduni.ranasinghe at uleth.ca<mailto:maduni.ranasinghe at uleth.ca>; Bowler, Bruce <Bruce.Bowler at mso.umt.edu<mailto:Bruce.Bowler at mso.umt.edu>>; Frederick, Ariel <ariel.frederick at umconnect.umt.edu<mailto:ariel.frederick at umconnect.umt.edu>>
Subject: Re: AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Perfect! Thanks so much, and thanks to Bruce for handling our samples, I will check in with you during the morning session of our workshop. Amy, please remind me in case I forget.
Thanks, -Borries

On Wed, Jul 19, 2023 at 9:24 PM Jason Zhu <jasonz13 at uci.edu<mailto:jasonz13 at uci.edu>> wrote:
Hi Bruce, Ariel, and Borries,

The peptide (F19Cha) should ship tomorrow (Thursday) morning and arrive before 10:30 am on Friday. I've attached the shipping label and instructions for how much water to add to make 1, 3, and 9 mM samples.

Best,
Jason

On Wed, Jul 19, 2023 at 11:59 AM James S. Nowick <jsnowick at uci.edu<mailto:jsnowick at uci.edu>> wrote:
Hi All,

The bottom line is that the absorbance of trifluoroacetate should be negligible with respect to the peptide. The peptide absorbs so strongly at 214 nm that you will need to go to longer wavelengths. Alternatively, I think interference optics might be a good option.

Best,
James

On Jul 18, 2023, at 7:57 PM, Jason Zhu <jasonz13 at uci.edu<mailto:jasonz13 at uci.edu>> wrote:

Hi Borries and James,

I tried running different TFA concentrations on the nanodrop and got very noisy UV-Vis spectra that I don't fully trust. I enabled the automatic baseline correction and automatic pathlength adjustment settings on the nanodrop which seems to have helped with the negative absorbance values that don't make physical sense. I will meet with James tomorrow morning and will work out the details of getting accurate absorbance values for both TFA and F19Cha then.

<image.png>

I plan on weighing out the peptides, labeling them, and shipping them by the end of the day Wednesday (tomorrow). I'll send an update once we figure out the ideal wavelength for the different concentrations we plan to run at each concentration and when the peptide is on its way.

For lyophilized powder, 4C should be adequate. F19Cha isn't prone to oxidizing from my experience working with it.

Best,
Jason

On Tue, Jul 18, 2023 at 2:38 PM Borries Demeler <demeler at gmail.com<mailto:demeler at gmail.com>> wrote:
Hi Bruce and Ariel:
We would like to have some samples shipped to U of Montana, and I would like to pick them up on Friday, if this is possible? The samples are lyophilized peptide samples and can probably be stored at 4C. I will be in Missoula on Friday  and Saturday, all day, our entire lab will actually be in Missoula for a vaccine development workshop with the CTM group. Thanks for helping again with the samples!!

James and Jason:
Regarding the sample shipment, you can overnight them as a lyophilized powder to:

University of Montana
Dept. of Chemistry &Biochemistry
c/o Bruce Bowler or Ariel Frederick
23 Campus Drive
Missoula, Montana 59812

Please weigh them out and indicate on your shipping list with what volume they need to be suspended to get the desired concentrations. Our loading volume will be 110 ul, and we would like to know the wavelength to use where we would get 0.6 OD in a 3 mm pathlength centerpiece, with no more than 0.3 OD of background absorbance from the buffer (when blanked against water). We can use different wavelengths for each sample to optimize the absorbance for each concentration.

The speed will be 60 krpm, so they should be measured in the An60Ti rotor, at 20C.

Please ship so they will be available for pickup by us on Friday this week. Please indicate the storage conditions - I assume for the lyophilized powder 4C is adequate?

Thanks, -Borries

James S. Nowick
Distinguished Professor
Department of Chemistry & Department of Pharmaceutical Sciences

Department of Chemistry
4126 Natural Sciences 1
University of California, Irvine
Irvine, CA 92697-2025

Phone:  (949)  824-6091
e-mail: jsnowick at uci.edu<mailto:jsnowick at uci.edu>
Faculty Web Page: http://tinyurl.com/jsnowick/<https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!N1pni7MvQC2b2B8UkuEuyJfTSy3SNOX8O3u82A0rJS42kQP5CMvnPCF331ntQaU-5VV5CG3ejXgg-gw0D2sQwB0-qA$>
Research Group Web Page: http://tinyurl.com/nowickgroup/<https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!N1pni7MvQC2b2B8UkuEuyJfTSy3SNOX8O3u82A0rJS42kQP5CMvnPCF331ntQaU-5VV5CG3ejXgg-gw0D2ux34kS7g$>
Pronouns: he/him/his

James S. Nowick
Distinguished Professor
Department of Chemistry & Department of Pharmaceutical Sciences

Department of Chemistry
4126 Natural Sciences 1
University of California, Irvine
Irvine, CA 92697-2025

Phone:  (949)  824-6091
e-mail: jsnowick at uci.edu<mailto:jsnowick at uci.edu>
Faculty Web Page: http://tinyurl.com/jsnowick/<https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!Pa-b8ObzdUvFuCSfCNJqhfvF9uZw9D0Dot6lNb2qzJbQwrgtD-kp20F44c3pcW9Gn9XoPbUSbTRH_n4$>
Research Group Web Page: http://tinyurl.com/nowickgroup/<https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!Pa-b8ObzdUvFuCSfCNJqhfvF9uZw9D0Dot6lNb2qzJbQwrgtD-kp20F44c3pcW9Gn9XoPbUS6KQo-ck$>
Pronouns: he/him/his

James S. Nowick
Distinguished Professor
Department of Chemistry & Department of Pharmaceutical Sciences

Department of Chemistry
4126 Natural Sciences 1
University of California, Irvine
Irvine, CA 92697-2025

Phone:  (949)  824-6091
e-mail: jsnowick at uci.edu<mailto:jsnowick at uci.edu>
Faculty Web Page: http://tinyurl.com/jsnowick/
Research Group Web Page: http://tinyurl.com/nowickgroup/
Pronouns: he/him/his

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