I am trying to do phenylalanine quenching studies for my peptide. It has 2 tyrosine and 2 phenylalanine and no tryptophan in its sequence. Its a transmembrane domain, so I want to find out whether its going into the membrane or not? I did phenylalanine quenching study in both acidic and neutral condition, and also in the presence of SDS micelles with excitation at 257 nm and emission from 270-450 nm. When I am plotting the graph F0/F Versus Q concentration, I am getting an upward curvature, indicating its showing both dynamic and static quenching. Could anyone please help me how to calculate Stern -Volmer constant for combined quenching? And what should be the minimum difference? Or should I have to do tyrosine quenching? Could anyone please suggest me in this?
Dear Hiasindh,
It is best to use a non-linear regression method if you want to avoid solving using the quadratic equation. Here I have attached an excel file that outlines some random data set, and a Stern-Volmer fit to the data, outlined as model. I have also described in orange how to use this spreadsheet. Once you complete the non linear regression using solver, you will obtain Ksv and Ka values.
Hope this helps with your work.
Ali
PS. If you are interested, there is a tolerance field within solver lowering which increases accuracy of Ksv and Ka estimates.
You would want to use the following equation:
I/I0=(1+kq*tau*[Q])(1+Ka[Q])
kq=biomolecular quenching rate constant
tau=excited state lifetime in the absence of quencher
Ka=association constant of the complex
Is this what you were looking for?
Ya. Am using this equation to calculate. So am plotting the graph between ((F0/F)-1)/[Q] Versus [Q], to find the Ksv and Ka constant. I read in some books that Slope is KsvKa and the intercept is Ksv+Ka. Am i right? But how to produce after plotting graph. Can you explain me with an example? And should i have to find the constant Ksv and Ka value separately?
Dear Hiasindh,
It is best to use a non-linear regression method if you want to avoid solving using the quadratic equation. Here I have attached an excel file that outlines some random data set, and a Stern-Volmer fit to the data, outlined as model. I have also described in orange how to use this spreadsheet. Once you complete the non linear regression using solver, you will obtain Ksv and Ka values.
Hope this helps with your work.
Ali
PS. If you are interested, there is a tolerance field within solver lowering which increases accuracy of Ksv and Ka estimates.
Hi Muhammad Ali,
I have plotted the graph F0/F against Q and inserted graph for that. I have also added solver in plug-in. But how did you put Model in Datasheet as well as in graph.
Also, after calculating using solver, how to obtain Ksv and Ka values. Could you please kindly explain me this? I have also added the F0 and F value in the datasheet.
Herewith am attaching my excel sheet. Please help me in solving this. Thanks in advance.
Thank you so much for your timely help.
I have calculated log(F0)/log(F) values and plotted against Arcylamide concentration.
Herewith, am attaching the excel sheet with slope value calculated. Kindly go through it and confirm me whether it is correct.
Am having one more doubt. Does slope directly equals to Ksv constant? or do we have to calculate Ksv value by dividing y-intercept by slope? Please do clarify my doubts.
Thanks in advance.
Hi Muhammad;
I saw your formula for the calculation of the stern volmer in both dynamic and static quenching occur. In your data set, you put some Quencher (Q) values without unit. What is the unit of quencher? or is it just proportion of concentration? It is because, it effect the result of calculation. Thank you for the answer.
Hi Dear Ashmi! did you finished your calculations? How you calculate KSV and Ka ? please do you have a spreadsheet, i also wanna do that.
I would also love to see the spreadsheet please, I have studied HSA with spectrofluorometry and I have also observed an upward curvature when plotting If0/If versus the concentration of my complex. I have calculated the binding constants with PSEQUAD but I would also love to compare this result with Ksv, Muhammad please help me, my email address is [email protected] thank you
Hi,
Herewith, am attaching the excel file sent by Muhammad.
But, I myself have doubt in that about the value in Model coulmn, how it was calculated and the ss value. I have mailed to Mohammed regarding the same, but no reply. If you do know how to calculate, please let me know.
Pl see the following reference
www.jh-inst.cas.cz/~fluorescence/support/Lectures/UFCH_fluor04.pps
What is reason the different values between Ka and Ksv?
Could anyone please suggest me in this?
Please be aware tha thte excel sheet posted Stern-Volmer.xlsx'' is incorrect, it should be I_0/I
Ya I agree with Marks. The plot should be in between F0/F and the concentrations of the quencher.
New to Stern-Volmer...My results is also an upward curve in F0/F-to-[Q] plot, which fits quadratic equation very well (y = 843.05x2 - 113.77x + 4.74, R² = 0.9989). So what should I do next to find Ka & Ksv?
F0/F= KaKsv[Q]2+(Ka+Ksv)[Q]+1= 843.05x2 - 113.77x + 4.74
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics