Susan and everyone, I should apologise for any confusion that I may have caused.
Rajiv actually asked his question a year ago, and I accidentally replied to it a year too late! It's an interesting question though Patrick On Wed, Sep 28, 2011 at 5:03 PM, 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 > > -- 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
