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Re: [ccp4bb] Thermofluor experiment |
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CCP4bb navigationCCP4bb <-- 2008 <-- March 2008 <-- 07 March 2008Subject: Re: Thermofluor experiment From: Brenda Patterson ucbccka {- at -} UCL {- dot -} AC {- dot -} UK Date: 2008-03-07 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 > 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 >> kornelius.zeth@tuebingen.mpg.de >> Tel -49 7071 601 323 >> Fax -49 7071 601 349 >> > CCP4bb navigationCCP4bb <-- 2008 <-- March 2008 <-- 07 March 2008 |
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