Hi all,
I have been using CCVJ dye for extrinsic fluorescence analysis to investigate tertiary structure of protein. The dye has been dissolved in DMSO which is of 20mM and further diluted to 5uM in de-ionized water in my final sample preparation. My fluorescence intensities are getting lower with increase in dye concentration in my final sample preparation.
What could be my final dye concentration for analysis?
Is it recommended to use water for final sample preparation?
Kindly suggest.
Please see the following papers for reference.
1: Hawe A, Sutter M, Jiskoot W. Extrinsic fluorescent dyes as tools for protein
characterization. Pharm Res. 2008 Jul;25(7):1487-99. doi:
10.1007/s11095-007-9516-9. Epub 2008 Jan 3. Review. PubMed PMID: 18172579; PubMed
Central PMCID: PMC2440933.
1: Ablinger E, Leitgeb S, Zimmer A. Differential scanning fluorescence approach
using a fluorescent molecular rotor to detect thermostability of proteins in
surfactant-containing formulations. Int J Pharm. 2013 Jan 30;441(1-2):255-60.
doi: 10.1016/j.ijpharm.2012.11.035. Epub 2012 Nov 28. PubMed PMID: 23200956.
Thanks Carlo. I have these papers, but I am confused with my sample preparation and data interpretation. I want to have a final confirmation to know what I am doing is right regarding the procedure and analysis.
CCVJ is sensitive to viscosity of the solution, when diluting your stock in water based buffer the viscosity of your solution is going to change. This doesn't matter much, you should have a baseline few thousand relative units and when doing a temperature scan you should clearly see a change in fluorescence, from which you can obtain a Tm. Please try doing one experiment and if anything is not clear come back with more specific questions.
Did you try other, more standard, dyes such as Sypro orange?
Remeber, DSF is a good screening technique for looking at protein unfolding under different buffer conditions. If you want to obtain accurate Tm (and much more) you should use DSC.
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