[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Tue Jul 25 17:18:42 MDT 2023

Hi Borries and Amy,

Here's the zoomed in spectra from the Nanodrop that you requested with the
10 mm absorbance in the 0-3 OD range. I also normalized the Y-axis to a 3
mm absorbance in that range in case it would be helpful to look at. I can
look into the dynamic and linear absorbance range of the instrument to see
how much we should trust these UV-Vis spectra.

Also, if the experiment works better with the 10 mm centerpieces for the
interference optics, I can provide more material as needed.
[image: image.png]
[image: image.png]

Best,
Jason

On Tue, Jul 25, 2023 at 4:06 PM Henrickson, Amy <amy.henrickson at uleth.ca>
wrote:

> 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
>
>
> 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 at uleth.ca.
>
> 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>
> 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>
> *Sent:* Tuesday, July 25, 2023 3:20 PM
> *To:* Borries Demeler <demeler at gmail.com>
> *Cc:* Jason Zhu <jasonz13 at uci.edu>; Henrickson, Amy <
> amy.henrickson at uleth.ca>; 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
>
>
> 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 at uleth.ca.
>
> 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> 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> 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> wrote:
>
> Wonderful! Thanks, Bruce!
> James
>
> On Jul 21, 2023, at 10:21 AM, Bowler, Bruce <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>
> *Sent:* Wednesday, July 19, 2023 9:26 PM
> *To:* Jason Zhu <jasonz13 at uci.edu>
> *Cc:* James S. Nowick <jsnowick at uci.edu>; Henrickson, Amy <
> amy.henrickson at uleth.ca>; demelerlab at biophysics.uleth.ca;
> maduni.ranasinghe at uleth.ca; Bowler, Bruce <Bruce.Bowler at mso.umt.edu>;
> Frederick, Ariel <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> 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> 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> 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> 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
> Faculty Web Page: http://tinyurl.com/jsnowick/
> <https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!N1pni7MvQC2b2B8UkuEuyJfTSy3SNOX8O3u82A0rJS42kQP5CMvnPCF331ntQaU-5VV5CG3ejXgg-gw0D2sQwB0-qA$>
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> Pronouns: he/him/his
>
>
> James S. Nowick
> Distinguished Professor
> Department of Chemistry & Department of Pharmaceutical Sciences
>
> Department of Chemistry
> 4126 Natural Sciences 1
> University of California, Irvine
> Irvine, CA 92697-2025
>
> Phone:  (949)  824-6091
> e-mail: jsnowick at uci.edu
> Faculty Web Page: http://tinyurl.com/jsnowick/
> <https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!Pa-b8ObzdUvFuCSfCNJqhfvF9uZw9D0Dot6lNb2qzJbQwrgtD-kp20F44c3pcW9Gn9XoPbUSbTRH_n4$>
> Research Group Web Page: http://tinyurl.com/nowickgroup/
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> 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
> Faculty Web Page: http://tinyurl.com/jsnowick/
> <https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!I66z_m6fiIkwmzYpyzCJLFt0VGQ-2-OlyMNthYqnMISq08OOrEdooCYoBTQ6TF9AS40WZ6ErdAjMV40tiWAEXTG8kOht$>
> Research Group Web Page: http://tinyurl.com/nowickgroup/
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> Pronouns: he/him/his
>
>
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