long question (3)

Question 1

two biomedical science students are investigating the activies of two proteins: protein X and protein Y, in cells in culture. their initial findings from immunofluorescence experiments suggest that when the cells are stimuated e.g. with a growth factor, protein X and protein Y co-localise in the same cellular compartment. the students now want to know whether the two proteins interact. describe how you would design an experiment using fluorescent-based techniques to investigate this. in your answer, you should also describe the expected results from your experiment according to whether protein X and Y do or do not interact

Question 2

how would you design an experiment with fluorescent-based techniques

Answer 2

• using fluorescence resonance energy transfer

Question 3

what is FRET

Answer 3

• an excited fluorophore of a high energy/short wavelength can transfer some of its energy to an acceptor fluorophore of a longer wavelength

Question 4

what is it used for

Answer 4

• protein-protein interactions
• ligand-receptor interactions
• protein dimerization
• protein conformational changes

Question 5

requirements for FRET

Answer 5

• two proteins tagged with different fluorophores
• the emission of protein X must overlap with the excitation of protein Y
• the proteins must be less than 10 nm apart

Question 6

what does FRET measures

Answer 6

• decreased fluorescence brightness of the donor
• increased fluorescence brightness of the acceptor from donor excitation
• decreased donor fluorescence lifetime

Question 7

FRET experiment

Answer 7

• protein X is tagged with ECFP (enhanced cyan fluorescent protein)
• protein Y is tagged with EYFP (enhanced yellow fluorescent protein)
• protein X is excited and if protein Y interacts it’ll get excited and emit fluorescence
• if not just protein X will emit fluorescence