Question

1) What is the great benefit of FLIM as compared to FRET? What is ratiometric FRET? Explain with diagrams. Which is the more precise method to use if you wanted to monitor the proteolytic processing of a genetically encoded fluorescent peptide reporter in vivo – explain why?

2) What was the major advance made in quantum dots for biological imaging discussed in class that involved the use of Protein A (SpA)? Explain the significance of the report and why it is useful? Describe how you would use these quantum dots in two separate experiments to observe in cells a histone protein for which you have a very good antibody and the epidermal growth factor receptor. Can you do these experiments in live cells or do the cells need to be fixed?

Answer 1

1. In Fluorescence Resonance Energy Transfer or FRET there is a donor and an acceptor molecule both of which are chromophores. If these two are present in close proximity, dipole- dipole coupling happens and then if the donor is exited (with apropropriate wavelength of light) then instead of getting emmision at a wavelength designated for the donor, but inturn we get emmision of the acceptor (the emmision of the donor excites the acceptor). But this can provide information Qualitatively. For example if you tag two proteins with two different chromophores, you will only get the information whether they interact wit each other or not, you wont get the information of how much they interact. To get this quantitative information, we need the help of fluorescence lifetime imaging or FLIM , with the help of this method we determine the distribution of the fluorescence lifetimes of a fluorophore at the different locations within the sample (With TCSPC Instrument) and the degree of interaction can also be obteined.

Ratiometric FRET Shows the ratio between the FRET signal and the donor intensity at donor excitation making small changes in FRET to get amplified, because donor signal correspondingly decreases as FRET signal increases. Ratiometric FRET is generally used in intramolecular biosensors.

For monnitering a proteolytic cleavage i will use a FRET-FILM system,beacuse that will not only provide me with the onformation that weather the proteolysis happens or not, but also how long it takes, how much it happens etc etc.