Hi, I did not see this particular instrument mentioned here so far: The FluoDia T70 from Photon Technology International, NJ, http://www.pti-nj.com/PlateReader/PlateReader.pdf
The whole instrument comes with its own thermal cycler and 96-well fluorescence plate reader with appropriate excitation and emission filters for sypro orange. The temperature control seemed quite sensitive and the analysis software was fine as well (accepts excel format). I was trying to test and set this equipment up for ligand binding assays for a particular integral membrane protein and some homologs with relatively weak binding affinities. It did not work for us, but I'm not sure whether that was related to protein degradation, some residual impurities from purification or due to variations in detergent micelle properties (and/or associations with hydrophobic portions of the protein, and competing with sypro orange binding) during the wide range of the temperature cycling. Regards, Debanu. -- Debanu Das, Stanford Synchrotron Radiation Laboratory, Menlo Park, CA. ------------------------------------------ -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Andreas Förster Sent: Wednesday, February 13, 2008 4:01 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Thermofluor experiment 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 > [EMAIL PROTECTED] > Tel -49 7071 601 323 > Fax -49 7071 601 349 >
