[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Tue Jul 25 18:41:03 MDT 2023

OK, this is very helpful. BTW, normalizing doesn't buy us anything, since
in the normalized sample only the 0-1 OD range is useful to us, which is
approximately the same as the 0-3 range for the 10 mm cuvette. Most
spectrophotometers are only linear in the range of 0-0.8 OD, at some
wavelengths maybe up to 1.2, rarely higher, only the best can get up to 1.5.

Remember, at 2 OD only 1 % of the incident light hits the detector, so
essentially no light comes through. It is comparing very dark to extremely
dark.

-Borries

On Tue, Jul 25, 2023 at 5:18 PM Jason Zhu <jasonz13 at uci.edu> wrote:

> 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
>>
>> Get Outlook for iOS
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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$>
>> Research Group Web Page: http://tinyurl.com/nowickgroup/
>> <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!N1pni7MvQC2b2B8UkuEuyJfTSy3SNOX8O3u82A0rJS42kQP5CMvnPCF331ntQaU-5VV5CG3ejXgg-gw0D2ux34kS7g$>
>> 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
>> 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
>> Faculty Web Page: http://tinyurl.com/jsnowick/
>> <https://urldefense.com/v3/__http://tinyurl.com/jsnowick/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!I66z_m6fiIkwmzYpyzCJLFt0VGQ-2-OlyMNthYqnMISq08OOrEdooCYoBTQ6TF9AS40WZ6ErdAjMV40tiWAEXTG8kOht$>
>> Research Group Web Page: http://tinyurl.com/nowickgroup/
>> <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!I66z_m6fiIkwmzYpyzCJLFt0VGQ-2-OlyMNthYqnMISq08OOrEdooCYoBTQ6TF9AS40WZ6ErdAjMV40tiWAEXW-STnfk$>
>> Pronouns: he/him/his
>>
>>
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