[3dem] Hexamer dissociate while freezing Cryo-EM grids

[Zhongwu Zhou]

Dear Ferrie,

The pH of UA is ~4. The common buffer for protein sample is pH ~6-8.
Depending on the settings of staining method, the lower pH can be
buffered!? Moreover, Uranyl acetate  acts as a fixative, preserving many
protein-protein interactions on a millisecond time scale. Thus, can you
give us some explosion of UA staining affecting the structure? the overall
structure or secondary structure?

best,

zhongwu

On Thu, Jun 4, 2020 at 3:34 PM Farzad <farzaad at gmail.com> wrote:

> Dear All,
>
> The other thing that should be considered is the low pH of UA staining
> agent. This may affect the structure.
>
> BR
>
> Ferrie
>
>
>
> On Thu, Jun 4, 2020, 22:28 Davide <dafloris at biophys.mpg.de> wrote:
>
>> Hi Umar
>>
>> you can find very useful advice in our publication:
>>
>> *Protein denaturation at the air-water interface and how to prevent it*
>>
>> https://urldefense.com/v3/__https://elifesciences.org/articles/42747__;!!Mih3wA!UzHoeUFE1ZlTNUXTnq9BnOBR4cui5h9guZQyocAQiVBGZOzPFzj8Zk-vviXh_YPYSA$ 
>> <https://urldefense.com/v3/__https://elifesciences.org/articles/42747__;!!Mih3wA!VfLdm8jk1oLaorfhmBpHU8SgDDDqoBFeugRuH6bNfz5IOsbAbVHiBNK7SqXAfdlwNw$>
>>
>> Best regards,
>>
>>
>> Davide
>> _________________________________________________
>>
>> Davide Floris
>> PhD student
>> Max Planck Institute of Biophysics
>> Structural Biology Department
>> Max-von-Laue Straße 3
>> 60438 Frankfurt am Main
>> Germany
>>
>> (L2.090): +49-69-6303-3037
>> (B2.205): +49-69-6303-3049
>>
>> On 4. Jun 2020, at 21:24, Umar Farook <umarfarook12 at gmail.com> wrote:
>>
>> Dear All,
>>
>> I would like to ask the community for suggestions on how to overcome the
>> issue of hexamer dissociating while freezing Cryo-EM grids, the negative
>> staining seems to be perfect hexamer at 0.1 mg/ml concentration. But the
>> sample seems to fall apart at 1.5 mg/ml in Cryo freezing, please advise,
>> thank you.
>>
>> Best Regards,
>> Umar
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