[Demelerlab] AUC Collaboration: Sizing Amyloid Beta derived Oligomers

[Borries Demeler]

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> 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> 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> 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> 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> 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
> Faculty Web Page: http://tinyurl.com/jsnowick/
> Research Group Web Page: http://tinyurl.com/nowickgroup/
> Pronouns: he/him/his
>
>
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