This paper on thermofluor is a good reference and if you have access to a real time PCR machine, different buffer systems, like the PACT screen can be evaluated within an hour to find out the buffer in which your protein is most stable. It gives the Tm of your protein and if you have a high fluorescence to start with, it means your protein is unfolded to start with.
Curr Protoc Mol Biol. <http://www.ncbi.nlm.nih.gov/pubmed/21472694#> 2011 Apr;Chapter 10:Unit10.28. The combined use of the Thermofluor assay and ThermoQ analytical software for the determination of protein stability and buffer optimization as an aid in protein crystallization. Phillips K<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Phillips%20K%22%5BAuthor%5D> , de la Peña AH<http://www.ncbi.nlm.nih.gov/pubmed?term=%22de%20la%20Pe%C3%B1a%20AH%22%5BAuthor%5D> . Best regards Shankar On Wed, Sep 28, 2011 at 11:03 AM, Linda Schuldt <lschu...@mb.au.dk> wrote: > Dear Raji, > > what exactly do you mean when you say the melting temperature is 45deg. > Did you only test one buffer, or did you test many buffers and 45deg is > the most stable one? If you have only tested one buffer you should run a > screen testing different buffer systems (pH) and e.g. NaCl concentration > and glycerol concentrations (or ligands, if your proteins binds any). Then > you identify the buffer which is stabilizing your protein the most. I have > seen big impacts on protein stability and crystallization when optimizing > my buffers like this. > > I think you should not only consider the melting temperature alone, but > also how the curve looks like. Do you get a high initial flourescence > (which often indicates partially unfolded protein or hydrophobic patches) > or do you have very low initial flourescence (which is a good sign for > compact protein). Another thing to look at is if your transition is sharp > (the steeper the better). Taking all this together you can judge if your > protein is happy or not. > > Hope this helps you! > > Linda > > Patrick Shaw Stewart wrote: > > I actually think you *can *make comparisons between different proteins. > > We > > heard a very nice talk by Jose Marquez about exactly this at the RAMC > > meeting recently. > > > > Basically, 45C seemed to be the dividing line. If your protein melts > > below > > this it's a bad sign for crystallization and may point to setting up your > > crystallization experiments at lower temperatures. > > > > Patrick > > > > > > > > On Thu, Sep 23, 2010 at 6:04 PM, Anastassis Perrakis > > <a.perra...@nki.nl>wrote: > > > >> ** > >> > >> Hello - > >> > >> The excellent paper of McCrary, uses differential scanning > >> calorimetry, which will give an absolute measure of thermostability. > >> > >> Using Thermofluor I would be afraid you can only assess the relative > >> thermostability of one protein in different conditions. > >> As your fluorescence reporter would interact differently with exposed > >> hydro[hobic patches in different proteins, I would be a bit more careful > >> in comparing the Thermofluor results between different proteins ... I > >> am not aware of anyone correlating differential scanning calorimetrywith > >> Thermofluor data, but I must admit I have not looked up that > >> literature recently. > >> > >> A. > >> > >> > >> On 23 Sep 2010, at 18:40, Philippe DUMAS wrote: > >> > >> > Le 23/09/2010 17:28, Raji Edayathumangalam a écrit : > >> > > >> > Raji > >> > I suggest having a look to this paper: > >> > McCrary et al. J. Mol. Biol. 264(1996) 784 > >> > where you will find an interesting study on protein stability and an > >> > interesting comparison with other proteins. > >> > Philippe Dumas > >> > > >> >> Hi Folks, > >> >> > >> >> Sorry for the pre-xtallo question; pre-xtallo right now, but hoping > >> >> to > >> >> take my protein the xtallo way one of these days! > >> >> > >> >> I am currently performing Thermofluor assays with my protein and the > >> >> results show that the Tm is ~45C. I am looking for some examples of > >> >> proteins and their melting temperatures so that I can gauge where my > >> >> protein falls in the spectrum of unstable-to-stably folded. For > >> >> example, the melting temperature of some forms of lysozyme is 73.8C > >> >> (very stable, I suppose). > >> >> > >> >> Just need a sense for whether my protein is considered unstable or > >> >> somewhat stable. Please could you share some examples. > >> >> > >> >> Many thanks. > >> >> Raji > >> >> > >> >> ----------- > >> >> Raji Edayathumangalam > >> >> Joint Research Fellow > >> >> Harvard Medical School/ > >> >> Brigham and Women's Hospital > >> >> Brandeis University > >> >> > >> > > >> > <McCrary-JMB264(1996)784.pdf><p_dumas.vcf> > >> > > > > > > > > -- > > patr...@douglas.co.uk Douglas Instruments Ltd. > > Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK > > Directors: Peter Baldock, Patrick Shaw Stewart > > > > http://www.douglas.co.uk > > Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 > > Regd. England 2177994, VAT Reg. GB 480 7371 36 > > > > > > > > > ******************************* > Dr. Linda Schuldt > Department of Molecular Biology > University of Aarhus > Science Park > Gustav Wieds Vej 10c > DK-8000 Århus C > Denmark >
