1. Please report this exercise as if you were writing up an experimental method. Using the equation below, calculate the emission spectra s for Donor and acceptor molecules with absorption and emission spectra given in the spreadsheet (forster radius data(BB0344).xls).
Where Q = quantum yield of D in the absence of A
2 = factor relating to orientation of D and A
n = refractive index of media (i.e solvent)
J() = degree of spectral overlap between D & A
And
where F() =emission spectra of the donor in the range
to +Δ
() = extinction coefficient of acceptor at (M-1 cm-1)
Assume that the molecules are randomly orientated and are embedded in an aqueous medium with refractive index (n) of 1.333. The quantum yield of the Donor is 0.6.
Note: You should calculate the integral using whichever numerical methodology you prefer and marks will not be deducted for this. Marks will be given for explanation of your methods and the correct result. Please provide working files with your finished report.
NO MARKS (0 marks) WILL BE AWARDED FOR STUDENTS WHO USE PRE-EXISTING WEB RESOURCES, WHICH AUTOMATICALLY CALCULATE THE FORSER RADIUS, TO COMPLETE THIS ASSIGNMENT.
2. Please report this exercise as if you were writing up an experimental analysis. Examine the fluorescence lifetime data for a pair of proteins which have been labelled with Donor and acceptor fluorophores in a FRET experiment given below.
a. Given a control lifetime (in the absence of acceptor) of 2.15 ns, use the data to describe the outcome of the experiment. In your answer you should consider how the fluorescence lifetime reports FRET and the localisation of this signal.
b. Using the histogram data in excel spreadsheet (lifetime histogram (BB0344).xls) calculate FRET efficiency for all data points and plot this data as a histogram. Use this data to expand on the hypothesis you described in a).
c. From the FRET efficiency data and assuming a Förster radius of 6 nm calculate the donor-acceptor separation for all data points and display this data as a histogram.
