[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Wed Jul 26 10:14:58 MDT 2023

Thanks Borries for explaining why the normalization doesn't buy anything
and that spectrometers have a non-linear range at high absorbance!

James, I ran this as a spectral scan so the absorbance may be a bit low to
get an accurate measurement. I reran the absorbance with a single point
measurement at 250 nm and got an absorbance of 2.35 which is in better
agreement with what I got when I measured the absorbance with a cuvette. I
agree that the absorption might be a bit high to be reading accurately. It
sounds like Sophie and Aysha have a plan to get the wavelengths needed to
use to get good data.

Best,
Jason

On Tue, Jul 25, 2023 at 5:50 PM James S. Nowick <jsnowick at uci.edu> wrote:

> Hi Jason,
>
> Was this done as a single point measurement on the NanoDrop, or with a
> spectral scan? If you did it as part of a scan, the pathlength would likely
> be 0.03 mm, which would lead to an absorbance value of 0.0053 before
> normalization to 1.0 cm. This absorbance is too low to get an accurate
> measurement. With a single-point measurement, the pathlength would probably
> be 1.0 mm, which would likely give a more accurate value.
>
> I am surprised by the difference between the two spectrophotometers, but
> 2.2 a.u. is a bit high to be reading accurately.
>
> James
>
>
>
>
> On Jul 25, 2023, at 5:29 PM, Jason Zhu <jasonz13 at uci.edu> wrote:
>
> As a control, I tried independently checking the absorption on both UV-vis
> spectrometers at 250 nm in a 10 mm cuvette on the same 1 mM peptide sample.
> According to the Nanodrop spectrum the absorbance should be ~1.75 but I got
> a bit higher values from both spectrometers which generally seem to be in
> decent agreement with each other. This means that the nanodrop might not be
> as accurate as the specifications state as Borries correctly suspected.
>
> New UV-Vis (UV-16600PC): 2.194
> Old UV-Vis (MultiScan Go) : 2.276
>
> I meet with James tomorrow afternoon and can discuss more with him on how
> to find the best wavelength to use before we run the experiment!
>
> Best,
> Jason
>
> On Tue, Jul 25, 2023 at 4:34 PM James S. Nowick <jsnowick at uci.edu> wrote:
>
>> Hi Borries,
>>
>> The NanoDrop is designed for checking small volumes of highly
>> concentrated solutions. It’s not designed to replace cuvette measurements,
>> but it is convenient for getting values within a few percent for samples
>> that are too small or too concentrated to be measured in a cuvette. We have
>> not checked the linearity. It sounds like repeating the measurements in
>> dilute solutions in a cuvette in your lab would be a good idea.
>>
>> What concentration of proteins do you typically run in your 10 mm cells?
>> I am guessing they are in the low micromolar range. Even with the higher MW
>> of proteins, I am guessing that there is a lot less material in solution
>> than our peptides.
>>
>> In past studies with peptides. For the 0.1, 0.3, and 0.6 mM solutions in
>> our 2014 JACS paper, you were able to do UV, working about 60 nm away from
>> the UV maximum. For our JACS 2007 paper, you did absorbance scans at 230 nm
>> for the 15 uM sample and and Rayleigh interference for the 1.5 mM and 9 mM
>> samples.
>>
>> Best,
>> James
>>
>> On Jul 25, 2023, at 3:47 PM, Borries Demeler <demeler at gmail.com> wrote:
>>
>> Hi James,
>> Hmmm, when I measure a typical protein solution suitable for AUC
>> measurements in a 10 mm pathlength cell, I get OD values ranging between
>> 0-1 absorbance units, not 0-600. This is where my confusion stems from. If
>> these numbers are indeed correct, I guess that just means that the sample
>> you measured was ~600 fold more concentrated than what I usually deal with,
>> and measured with a pathlength of 0.3 mm? That being said, I would question
>> the accuracy of a measurement made with a 0.03 cm pathlength, since even a
>> tiny error in the pathlength would have very large error effects. Indeed,
>> one of our rules in the lab is to always repeat absorbance measurements
>> because frequently we get samples measured with a nanodrop-like device that
>> are completely unusable because their concentrations are totally off. Have
>> you checked the dynamic range of this instrument? If so, what is the linear
>> absorbance range? At any rate, I should expect about 50 OD at 230 nm for a
>> 9 mM sample in a 10 mm cuvette, am I reading this correctly?
>>
>> Regards, -Borries
>>
>> On Tue, Jul 25, 2023 at 3:20 PM James S. Nowick <jsnowick at uci.edu> wrote:
>>
>>> 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!J5JZO4wHinfruDdUmtXfjcfpYgzfPuuMMecjmpQM5QnjtRE67du0N5KFiHhS5nMU3VR1geY3XBsfed0$>
>>> Research Group Web Page: http://tinyurl.com/nowickgroup/
>>> <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!J5JZO4wHinfruDdUmtXfjcfpYgzfPuuMMecjmpQM5QnjtRE67du0N5KFiHhS5nMU3VR1geY3ZHBOj1U$>
>>> 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!OPh_Ennt78PkI05SaBGsdf52XxFs-a0s2_cH9Y61bD2MjqlkK77UWb_c-y3Ns2ioydb5MYqohdYbkdVthg$>
>> Research Group Web Page: http://tinyurl.com/nowickgroup/
>> <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!OPh_Ennt78PkI05SaBGsdf52XxFs-a0s2_cH9Y61bD2MjqlkK77UWb_c-y3Ns2ioydb5MYqohdbMVO-T2g$>
>> 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/
> Research Group Web Page: http://tinyurl.com/nowickgroup/
> Pronouns: he/him/his
>
>
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