Re: [ccp4bb] Thermofluor assay

I've tried to post my reply to this quite a few times but as best I can tell (and my friends can tell from their CCP4bb subscriptions) I have been foiled by jiscmail every time. Hopefully this will go through. And hopefully I'm not reposting stuff I already sent. Here we go: Okay, the consensus he

Re: [ccp4bb] Thermofluor assay

There is an alternative method that does not rely on hydrphobic interaction of dye with the protein interior: it relies instead on reaction between fluorogenic dye and interior cysteine residues of the protein. When protein melts these Cys residues become exposed, react with the dye and generate fl

Re: [ccp4bb] Thermofluor assay

Hi Theresa, Check if this works in your case http://www.sciencedirect.com/science/article/pii/S0969212608000609 good luck !! On Sat, Apr 12, 2014 at 4:38 PM, Theresa Hsu wrote: > Dear all > > Does anyone has experience with Thermofluor assay to find the substrate > transported/binding by a mem

Re: [ccp4bb] Thermofluor assay

Dear Theresa,, , I would not advice to use thermofluor with membrane proteins as thefluorescent dye used in the assay binds to exposed hydrophobic patches of unfolding proteins. Therefore the presence of detergents would cause much unwanted fluorescence which would likely swamp your signal of in

Re: [ccp4bb] Thermofluor experiment

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

Re: [ccp4bb] Thermofluor experiment

ECTED] 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, seco

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 dr

Re: [ccp4bb] Thermofluor

yes indeed, the Biorad MiniOpticon is the cheapest so far. Next one on my list is the Applied Biosystems StepOne. Can anybody comment on the latter machine (not the more expensive StepOnePlus)? The machines proposed like Roche LC480, Stratagene Mx3005p or Biorad iQ5 are far beyond 30 k€ list p

Re: [ccp4bb] Thermofluor experiment

Kornelius, have a look at the following paper: Yeh AP, McMillan A, Stowell MH.

Re: [ccp4bb] Thermofluor

Hei Jeroen, Quite recently we got BioRad MiniOpticon system for the thermoflor based assays. I think this is the least expensive RT-PCR on the market but it performs very well in our experiments. It can excite the samples between 470 and 505 nm and one of the detection channel covers the

Re: [ccp4bb] Thermofluor

Hi Jeroen, We just bought a Stratagene Mx3005p for the Thermofluor method (also known as differential scanning fluorimetry). This was after talking to Martin, among others, he he... We haven't had it long and did our first experiments last Friday, but it produced good results straight awa

Re: [ccp4bb] Thermofluor

Hi Jeroen, We used a BioRad iCycler iQ for the measurements in "Thermofluor-based high-throughput stability optimization of proteins for structural studies." Ericsson, et. al. Analytical Biochemistry (2006) 357:289-298. The wavelengths for excitation and detection were ~490 and ~575 nm, resp