[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

James S. Nowick jsnowick at uci.edu
Tue Jul 25 17:33:05 MDT 2023 

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 <mailto: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 <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/ <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/
Research Group Web Page: http://tinyurl.com/nowickgroup/
Pronouns: he/him/his

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