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

[ccp4bb] Thermofluor assay

Dear all Does anyone has experience with Thermofluor assay to find the substrate transported/binding by a membrane protein? My protein does not have any similar structures and the substrate suggested by sequence analysis is not being transported in proteoliposome. I know ITC is good but I am

Re: [ccp4bb] Thermofluor assay

of interest. However, I have never personally tried it with a membrane protein . Best,, , D From: Theresa Hsu [theresah...@live.com] Sent: 12 April 2014 09:38 To: ccp4bb Subject: [ccp4bb] Thermofluor assay Dear all Does anyone has experience

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 theresah...@live.com wrote: Dear all Does anyone has experience with Thermofluor assay to find the substrate

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

[ccp4bb] thermofluor - Thermal Shifts - Tm and Delta Tm

Hello, we want to use the thermofluor thermal shift assay to screen for buffers that our proteins are happy in. Reading around the subject, I am a bit unclear as to what to be looking for with regard to a stabilizing affect. Initially, we thought, the higher the Tm, then the more stabilizing

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

[ccp4bb] Thermofluor again

Hi again, Martin pointed out to me that my description of the BioRad machine was in fact of an older model from ca. 2003 which indeed required manual filter changes. The iQ5 has multiplexing capability just like the Mx3005p. I'll leave further discussions to the experts ;-) Derek

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

[ccp4bb] Thermofluor experiment

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

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

Re: [ccp4bb] Thermofluor experiment

Kornelius, have a look at the following paper: Yeh AP, McMillan A, Stowell MH. http://www.ncbi.nlm.nih.gov/pubmed/16552147?ordinalpos=1itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

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

Re: [ccp4bb] Thermofluor experiment

] 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

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,

[ccp4bb] AW: [ccp4bb] Thermofluor

@JISCMAIL.AC.UK Betreff: [ccp4bb] Thermofluor Sorry for the off-topic but can somebody recommend highly a sensitive RT-PCR machine for the thermofluor experiment (sypro orange). That would imply excitation below 500 nm (ideally 470) and detection at about 570 nm, right? I know several simple machines have

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

[ccp4bb] Thermofluor

Sorry for the off-topic but can somebody recommend highly a sensitive RT-PCR machine for the thermofluor experiment (sypro orange). That would imply excitation below 500 nm (ideally 470) and detection at about 570 nm, right? I know several simple machines have a problem with the 570 nm...