[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

Tue Jul 18 15:06:17 MDT 2023

Hi Borries,

We have separate instruments to go with cuvettes. The Nanodrop is ideal for precious samples, particularly at higher concentrations.

James

> On Jul 18, 2023, at 2:03 PM, Borries Demeler <demeler at gmail.com> wrote:
> 
> Yes, thanks, James! I heard from someone who said you could get longer pathlength cuvettes for the Nanodrop device (maybe a different manufacturer?). We prefer to use a 1 cm pathlength benchtop UV-vis spec, but in this case the 1 cm pathlength would give even worse results. The reason this is an issue is because the light doesn't enter the cuvette perpendicularly, but at an angle. It is probably optimized for water or aqueous solutions. If you trace the optical track you can see that a high RI will actually focus the light on the center of the exit side, leading to higher intensity than was observed in the baseline scan.
> 
> -Borries
> 
> On Tue, Jul 18, 2023 at 2:57 PM James S. Nowick <jsnowick at uci.edu <mailto:jsnowick at uci.edu>> wrote:
>> Thanks, Borries. I hadn’t known that RI effects could be significant in such dilute solutions (0.1%).
>> 
>> For the Nanodrop, there is no cuvette, and the path length is 0.030-1.0 mm. I am attaching a description, in case you are interested.
>> 
>> Jason: For TFA, please do a cuvette measurement blanked against water.
>> 
>> James
>> 
>>> On Jul 18, 2023, at 1:46 PM, Borries Demeler <demeler at gmail.com <mailto:demeler at gmail.com>> wrote:
>>> 
>>> James,
>>> the negative absorbance is a result of chromatic aberration, which is linked to the refractive index of the TFA. Such errors are amplified at low wavelengths. One way to minimize it is to use the shortest possible pathlength cuvette. Regardless, we need to know the background absorbance of TFA in water (blanked against water). Please let me know if this was already a short pathlength cuvette, or if you used a 1 cm cuvette for this.
>>> 
>>> -Borries
>>> 
>>> On Tue, Jul 18, 2023 at 2:35 PM James S. Nowick <jsnowick at uci.edu <mailto:jsnowick at uci.edu>> wrote:
>>>> The negative absorbance of 0.1% TFA puzzles me too, as it does not make physical sense. I suggest you check again in a cuvette in the regular UV-Vis with water as a blank, Jason.
>>>> 
>>>> I am also puzzled by the falloff of the absorbance of the peptide at 200 nm and the negative peak below 200. My understanding is that the amide bond chromophore absorbs below 200, as well as above. It may be worth repeating the measurement. 
>>>> 
>>>> These discrepancies could be explained if the Nanodrop was not properly cleaned before running the blank and residual contamination of the blank resulted in absorbance.
>>>> 
>>>> James
>>>> 
>>>>> On Jul 18, 2023, at 1:21 PM, Borries Demeler <demeler at gmail.com <mailto:demeler at gmail.com>> wrote:
>>>>> 
>>>>> I suspect that the negative absorbance of TFA is a problem with chromatic aberration on your nanodrop device. This may be related to a high refractive index of TFA that distorts your measurement, if you got this result from a 1 cm cuvette you may want to try with a shorter pathlength to get a more accurate measurement. A negative absorbance doesn't make any sense. Do you have a trace from 220-280 nm for TFA? It would be good to see multiple concentration of TFA in water.
>>>>> Thanks, -Borries
>>>>> 
>>>>> 
>>>>> 
>>>>> On Tue, Jul 18, 2023 at 1:59 PM Jason Zhu <jasonz13 at uci.edu <mailto:jasonz13 at uci.edu>> wrote:
>>>>>> Hi Borries,
>>>>>> 
>>>>>> Apologies for the belated response, the purifications took a bit longer than expected.
>>>>>> 
>>>>>> I tried adding Tris (at pH 6.0 and pH 9.0) to 1 mM F19Cha and observed salting out at 10-50 mM Tris so it is not a viable additive to prevent non-ideal effects unfortunately. 
>>>>>> 
>>>>>> I ran nanopure water (blank), nanopure water with 0.1 % TFA (ca. 10 mM) and 1 mM F19Cha dissolved in nanopure water on our nanodrop. I see a slight negative peak when 0.1% TFA is added (run 1) and that this peptide absorbs strongly even at 1 mM F19Cha (run 2) on our nanodrop. 
>>>>>> <image.png>
>>>>>> I have some purified F19Cha (~24 mgs) that is ready for shipping. I can weigh out the peptides and write out the corresponding volumes of solution to add for our AUC studies. What would be the best address to ship to and when should I ship these peptides? 
>>>>>> 
>>>>>> Best regards,
>>>>>> Jason
>>>>>> 
>>>>>> On Sun, Jul 9, 2023 at 1:23 PM Borries Demeler <demeler at gmail.com <mailto:demeler at gmail.com>> wrote:
>>>>>>> Hi Jason,
>>>>>>> thanks for the PDB files, as I suspected we need new definitions for the various non-canonical AAs. I get errors when running SOMO on the undefined residues. I asked Emre from our group to suggest a way how to deal with this. Presumably someone needs to edit the definitions for the unknown residues. I'll give you some updates when I have them.
>>>>>>> 
>>>>>>> Regards, -borries
>>>>>>> 
>>>>>>> On Thu, Jul 6, 2023 at 11:37 PM Jason Zhu <jasonz13 at uci.edu <mailto:jasonz13 at uci.edu>> wrote:
>>>>>>>> Dear Borries and the Demeler lab, 
>>>>>>>> 
>>>>>>>> As a follow-up to our conversation today, I've attached my slides, PyMOL models for the crystallographic F19Cha hexamer and the proposed NMR dodecamer based on a structural analog (PDB ID: 5v63). I broke up the subunits of the crystallographic hexamer and proposed NMR dodecamer into separate pdb files to make them easier to work with. 
>>>>>>>> 
>>>>>>>> In the meantime, I'll try adding Tris to my peptide, running samples on the nanodrop, purifying more F19Cha and will keep you posted. 
>>>>>>>> 
>>>>>>>> Thanks again for setting up this meeting and having so many members of your lab attend. Please feel free to reach out if there's anything you need from our end. I look forward to working with you all! :)
>>>>>>>> 
>>>>>>>> Kind regards,
>>>>>>>> Jason
>>>> 
>>>> 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!OaZpHCAnILdrNJ-c9ecnQhoTxB179u3xmsGmuq1i3Y3qB_7VlREsyPnn_em1QixpJ7GlHchDodx9tIE$>
>>>> Research Group Web Page: http://tinyurl.com/nowickgroup/ <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!OaZpHCAnILdrNJ-c9ecnQhoTxB179u3xmsGmuq1i3Y3qB_7VlREsyPnn_em1QixpJ7GlHchDQDMsieo$>
>>>> 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!NtSCZ1vvUUzXpnnwjbI53c5l0AY3PEREMjBEtvP6otXId0xggidzib2nG9wGrwpGGG_g78EAufcbLl0$>
>> Research Group Web Page: http://tinyurl.com/nowickgroup/ <https://urldefense.com/v3/__http://tinyurl.com/nowickgroup/__;!!CzAuKJ42GuquVTTmVmPViYEvSg!NtSCZ1vvUUzXpnnwjbI53c5l0AY3PEREMjBEtvP6otXId0xggidzib2nG9wGrwpGGG_g78EAeUOQHhs$>
>> 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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