Anita, In terms of the basics:Thermodynamic stability of a protein is related to Gibbs Free Energy of unfolding. The Gibbs Free Energy is made of temperature, enthalpy and entropy (Delta G = Delta H - T Delta S). At Delta G equivalent to zero, the concentration of folded protein is equivalent to the unfolded protein. Thus, proteins are most stable at conditions where their melting temperature (Tm) is highest. Protein solubility and stability are prerequisites for subsequent biochemical and biophysical analysis and characterization. The preparation of concentrated, soluble, and stable protein sample can often be a difficult task as proteins aggregate, precipitate, or denature. Protein stability and solubility is directly correlated to temperature, pH, buffer composition, salt composition, additives, and ligands. The fluorophore ( ANS, SYPRO) has a high quantum yield in a low dielectric medium. Protein unfolding exposes the hydrophobic core corresponding to the low dielectric medium, and the fluorophore is quenched in the solution. Melting a proteins internal amino acid side chains is a first order phase transition associated with absorption heat. The Differential Scanning Fluorimetric (DSC, or Thermofluor) assay gives a direct measurement of a proteins melting temperature (Tm).In terms of Applications:The Thermofluor assay is used for the optimization of solution for protein stabilization. This is utilized for protein preparations and biochemical assays, crystallization, as well as optimization of ligand binding affinity, including drug screening, a measure of protein cooperatively, and to probe function. In terms of Extrinsic Dyes:SYPRO Orange is by far the most popular, along with ANS as they both are utilized to bind to the hydrophobic core of the soluble protein. CPM has been recently utilized to study membrane proteins as it binds to buried cysteines. I would look at the paper by Stevens et al 2008, and for high throughput, I would look at the utilization of CPM in the presence of detergents by Fan et al 2011. In terms of limitations, there are advantages and disadvantages:Advantages include the small of quantity of protein needed, the low concentration needed, the reproducibility, and the possibility of simultaneous screening of multiple conditions. For disadvantages, the interactions between the dye chosen and other compounds may mask stabilization or cause artifacts, as well as difficult interpretation effects of oligomeric proteins that show multi-phasic unfolding characteristics. Also, most dyes are not applicable to conditions comprising hydrophobic additives such as detergents that are necessary for membrane proteins (Hence, applications utilizing CNS). I hope this helps, good luck with your studies!Sincerely,lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA Peking UniversityThe College of Life SciencesBeijing, China
Date: Thu, 19 Jul 2012 11:00:28 -0400 From: jubo...@jhsph.edu Subject: Re: [ccp4bb] off topic Thermal shift assay To: CCP4BB@JISCMAIL.AC.UK Yes that is very true. But I assume true membrane proteins exclude high throughput :-) However the cytoplasmic part might be fine in this case and then I would just go for Sypro Orange. Jürgen On Jul 19, 2012, at 10:39 AM, Edwin Pozharski wrote:My understanding is that the advantage of the thermofluor assay is that you can test many conditions rapidly unless of course you have some kind of high throughput CD spectrometer in mind. > If you have a CD available (not the one with music on it) you don't need a > dye just sufficient protein and you can thermal denature your protein > assuming it contains some secondary structure elements. > > Jürgen > > > On Jul 19, 2012, at 4:26 AM, anita p wrote: > > Hi All, > I want to use a thermofluor for the thermal shift assay. My proteins are > cytoplasmic truncations of membrane protein. I have read about ANS, > sypro-orange and CPM. Which is the once that is popularly used by the > crystallographers for condition optimization for crystallization ?? > > I have read that it sypro orange is not good for hydrophobic proteins and > CPM can't be used with DTT or bME in the buffer. > I am a bit confused . > Please help > thanks in advance > Anita > > ...................... > 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://lupo.jhsph.edu > > > > > -- Edwin Pozharski, PhD University of Maryland, Baltimore ...................... 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://lupo.jhsph.edu
