Re: [ccp4bb] Protein melting temperatures
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.nlwrote: ** 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.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein melting temperatures
For what it's worth, we've been using thermofluor to compare the 'apparent' melting points of enzymes with their thermal stability measured as inhibition of their respective reactions by elevated temperature. The data so far make sense - the differences in apparent enzyme Tm (using the same conditions as the reaction mix!) match the differences in the half-inhibition T. Not the absolute number,though (which is not unexpected givn the different kinds of measurements involved). So I'd say thermofluor is reasonably good at comparing different proteins. Qualitatively at least. Artem On Wed, Sep 28, 2011 at 6:25 AM, Patrick Shaw Stewart patr...@douglas.co.uk 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.nlwrote: ** 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.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] Protein melting temperatures
We have proteins that melt at 60˚C but they don't crystallize. According to your 45 degree rule we should have crystals, what are we doing wrong ? Jürgen On Sep 28, 2011, at 10:05 AM, Artem Evdokimov wrote: For what it's worth, we've been using thermofluor to compare the 'apparent' melting points of enzymes with their thermal stability measured as inhibition of their respective reactions by elevated temperature. The data so far make sense - the differences in apparent enzyme Tm (using the same conditions as the reaction mix!) match the differences in the half-inhibition T. Not the absolute number,though (which is not unexpected givn the different kinds of measurements involved). So I'd say thermofluor is reasonably good at comparing different proteins. Qualitatively at least. Artem On Wed, Sep 28, 2011 at 6:25 AM, Patrick Shaw Stewart patr...@douglas.co.ukmailto:patr...@douglas.co.uk 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.nlmailto: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.pdfp_dumas.vcf -- patr...@douglas.co.ukmailto:patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.ukhttp://www.douglas.co.uk/ Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034tel:1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://web.mac.com/bosch_lab/
Re: [ccp4bb] Protein melting temperatures
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.nlwrote: ** 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.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas 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) 148-864-9090US 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
Re: [ccp4bb] Protein melting temperatures
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 Khttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Phillips%20K%22%5BAuthor%5D , de la Peña AHhttp://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.nlwrote: ** 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.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas 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) 148-864-9090US 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
Re: [ccp4bb] Protein melting temperatures
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.nlwrote: ** 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.pdfp_dumas.vcf -- patr...@douglas.co.ukDouglas 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) 148-864-9090US 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.ukDouglas 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) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
[ccp4bb] Protein melting temperatures
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
Re: [ccp4bb] Protein melting temperatures
There is a nice paper Comput Biol Chem. 2009 Dec;33(6):445-50. Epub 2009 Oct 20. Predicting melting temperature directly from protein sequences. Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N. They have a list of 35 different proteins with their Tms with the references from where they obtained their data. Hope this aids in your work. Dan
Re: [ccp4bb] Protein melting temperatures
Not sure whether this is the kind of information you are looking for: The protein with PDB-ID 1ofc had a melting temperature of 37deg (from CD), which was supported by the fact that it did not express in E.coli at that temperature. At 20deg it expressed to about 60mg / (liter LB), could be concentrated to more than 100mg/ml, crystallised at room temperature and diffracted to 1.9A. The initial purification steps were done at 4deg, but I guess that's generally good advice anyhow. So maybe you don't need to worry too much, because stability is probably not the same as thermal stability. Cheers, Tim On Thu, Sep 23, 2010 at 11:28:20AM -0400, Raji Edayathumangalam wrote: 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 -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Protein melting temperatures
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.pdfp_dumas.vcf
Re: [ccp4bb] Protein melting temperatures
I agree that you can't take two unrelated proteins and expect their Thermofluor Tms will be correlated with CD/DSC values. We've done quite a bit with point mutants, and it works well for that (see an example in our paper below). Also note that the dye is a perturbant the reduces the apparent Tm at higher concentrations, and of course that the whole point of using Thermofluor to help find Xtal conditions is that the apparent Tm is sensitive to buffer, ligands, etc. Tom http://www.chemistry.ohio-state.edu/~magliery/pdfs/LavinderMagliery2009JACS.pdf On 9/23/2010 1:03 PM, Anastassis Perrakis 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.pdfp_dumas.vcf -- Thomas J. Magliery, Ph.D. Assistant Professor Department of Chemistry Department of Biochemistry The Ohio State University 1043 Evans Laboratory 100 West 18th Ave. Columbus, OH 43210-1185 (614) 859-5743 phone (Google Voice) (614) 292-1685 fax magli...@chemistry.ohio-state.edu http://www.chemistry.ohio-state.edu/~magliery
