Order Basic Fluorescence Techniques Assignment

Order Basic Fluorescence Techniques Assignment
3.1 Fluorescent labels are designed in such a way that the fluorescent quantum yield is as high as possible and that the fraction of triplet state
remains as small as possible upon excitation. Some time constants of two
different fluorescent labels are given below:
Label A Label B
Radiative S1 ® S0 transition
(fluorescence):
τFl = 5 ns τFl = 5 ns
Nonradiative S1 Î S0 transition
(internal conversion):
τIC ˆ 25 ns τIC ˆ 25 ns
S1 Î T1 intersystem crossing: τISC = 100 ns τISC = 20 ns
Figure 3.34 Luciferase from the firefly Renilla reniformis catalyses the oxidation of coelenterazine to oxy-coelenterazine in the presence of O2. In contrast to luciferase from the firefly Photinus pyralis it does not require the presence of ATP or Mg2 , and results in photons at ∼480 nm.
100 3 Basic Fluorescence Techniques
Radiative T1 ® S0 transition
(phosphorescence):
τPh = 2 s τPh = 2 s
Order Basic Fluorescence Techniques Assignment
(internal conversion):
τT!S
ISC ˆ 20 μs τT!S
ISC ˆ 50 μs
Which dye is the better fluorescent label? Why? What other properties
that can not be deduced from the given time constants are also important
for a fluorescent dye to be a good label for biomolecules?
3.2 You have identified potential binding regions at the surface of two proteins that form a stable receptor–ligand complex. To check if they indeed
bind together at these regions you plan to use a Förster energy-transfer
experiment. Where would be the ideal labelling sites at the proteins?
Would you rather use amino group reactive labels or thiol group reactive
labels? How would you achieve a specific labelling?
3.3 You want to observe by fluorescence detection where in a cell a certain protein is the most abundant. The protein is expressed by the cell itself. It turns
out that it is impossible to specifically attach fluorescence labels to these
proteins within the cell. What alternative approach is possible to observe
fluorescence that indicates the presence of these proteins in the cell?
Explain how you can purify proteins that contain sequences of histidines
from a mixture of different proteins.
3.5 Assume you have a solution of 1 nM streptavidin. What concentration of
biotin is necessary that the probability that a single biotin binds to one
streptavidin subunit is at least 50%?