Hi,
I am interested in using the thermofluor to assess the stability of my protein
in different buffers. Can anyone recommend a vendor that supplies buffer
screens, possibly in 96 well format?
Not crystallization buffers, just ordinary storage buffers.
Thanks
brenda
Quoting Andreas Förster <[log in to unmask]>:
> Dear Kornelius,
>
> I found the idea of doing Thermofluor on membrane proteins really
> intriguing - for identifying the best buffer and detergent, secondary
> detergents, even for checking crystallization drops that stayed
> clear. (This latter experiment should theoretically be possible with
> large drops, even though you'd be working very near the limits
> claimed in the publications.)
>
> In the end, I didn't find the method too useful because of noise
> issues due to detergent and exposed hydrophobic portions of the
> protein. I always felt that, in order to get reasonable signal, I'd
> have to use protein at unreasonable concentrations.
>
> The method works much better for soluble protein, though I can't tell
> you a success story where it led to crystallization that seemed
> impossible before.
>
>
> Andreas
>
>
> Kornelius Zeth wrote:
>> Dear all,
>>
>> a question very related to the discussion before. I have been
>> reading the papers about the thermofluor experiment with great
>> interest. I wonder what people think about the underlying
>> principles/ideas and the success that the method yielded in their
>> own labs for crystallization or related purposes?
>>
>> Has anybody used this method with membrane proteins in order to find
>> out the stability of the protein in the presence of a second
>> detergent?
>>
>> Is the method limited to this certain dye (sypro orange)?
>>
>> Have a nice day
>>
>> Kornelius
>>
>> P.S.: I will make a summary of all opinions.
>>
>> ----------------------------------------------
>> Kornelius Zeth
>> Max Planck Institute for Developmental Biology
>> Dept. Protein Evolution
>> Spemannstr. 35
>> 72076 Tuebingen, Germany
>> [log in to unmask]
>> Tel -49 7071 601 323
>> Fax -49 7071 601 349
>>
>
|