Why not thermal denaturation in the presence of Sypro Orange and a realtime PCR machine ?
Crowther et al. Use of thermal melt curves to assess the quality of enzyme preparations. Anal Biochem (2010) vol. 399 (2) pp. 268-275 Or (shameless advertisement): Hain et al. Structural characterization and inhibition of the Plasmodium Atg8-Atg3 interaction. Journal of Structural Biology (2012) vol. 180 (3) pp. 551-62 2.9.2. Thermal shift assay Mutant and wild type proteins were tested at 1 mg/mL in a reaction containing 30 lL of protein and 1 lL of a 1:30 dilution of SYPROÒ orange dye. Fluorescent measurements were done in triplicates in a CFX96 thermal cycler (BioRad) from 20 to 80 °C over a period of 60 min and the melting temperature was determined from the maximum of the first derivative of the melting curve. You will need less protein compared to the CD melt. Jürgen On Mar 20, 2013, at 8:56 PM, Kavestri wrote: Hi Harsh Something like sodium borate at pH 9.0 could be an alternative to phosphate buffers. If you are looking at thermal unfolding above 220nm, then the choice of buffer is less critical as many buffers and additives are problematic only below 200nm. If your samples require high salt concentrations, I routinely use NaF as an alternative. It is transparent in the wavelength range relevant for far UV CD spectral collection. Kavestri Y. Kingston Laboratory - Structural Biology Group University of Auckland On 20/03/2013, at 7:59 PM, Harsh Bansia <spideysp...@gmail.com<mailto:spideysp...@gmail.com>> wrote: Sorry for a simple and non-CCP4 question. I have determined the structures of three different mutants of a thermostable protein by X-ray crystallography method. I feel that Mg2+ has a role in protein stability. So I want to perform a thermal denaturation study by CD spectroscopy both in presence and absence of Mg2+ ion. In this regards, what should be the suitable buffer for CD studies? May I use PBS buffer ? Since phosphate sequester divalent cations like Mg2+. Is it advisable to use PBS buffer. If so, what is maximum concentration of Mg2+ ion that can be used say e.g. 5 mM? My protein was in Tris buffer and lyophilized and have theoretical pI =4.56 and maximum activity at pH 8.4. Thanking you in advances, harsh ...................... 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
