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1) Protein W is a membrane-spanning protein in the nucleus that is attached to the nuclear lamina. If you want to perform a FRAP experiment on Protein W and you have a GFP-tagged version of Protein W, what do you see following bleaching of the GFP signal with a laser?

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Jarrod Robel
Jarrod RobelLv2
28 Sep 2019

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Related questions

You have purified protein you call MAP15. MAP 15 binds totubulin that is GTP-bound tubulin and does not bind to tubulin thatis GDP-bound. You genetically engineered cells to express a greenfluorescent protein (GFP) –tagged version of MAP15. This GRP-MAP15fusion protein does not appear to disrupt the function of MAP15.You use a red fluorescent dye to label the microtubules so that youcan visualize GFP-MAP15 and the microtubules at the same time usingfluorescence microscopy (Note that the red dye does not appear toaffect microtubule structure and function).

A. If you were to polymerize some red-labeled microtubules in adish, would you expect GFP-MAP15 to be associated with one or bothends of a microtubule, with the middle of the microtubule, or beevenly distributed throughout the microtubule? Explain.

B. You examine the red microtubules and GFP-MAP15 in a cell ininterphase. Do you think that you will see GFP-MAP15 associatedwith all microtubules? Explain.

C. If you were to take time-lapse images in which you visualizeonly GFP-MAP15 (and not microtubules), what would you expect tosee? Describe what you think would happen to GFP-MAP over time withrespect to its localization inside of this cell.

1. This question about FRAP will require some thinking and you maywant to draw a concept map. You genetically engineer your membraneprotein so that GPF is fused to itsC-terminus. This construct can get to the membrane on itsown and give fluorescence. You also make a second version of thesame protein, by fusing RFP to theN-terminus of the protein (RFP = red fluorescent protein).This protein can also get o to the membrane on its own and givefluorescence. Now you transfect one group of cells with theGFP-tagged protein, and a different group of cells with theRFP-tagged protein and you conduct FRAP experiments. You obtain thefollowing two FRAP graphs:

a. Label the X and Y axes for each graph and put units and put somearbitrary but reasonable numbers.

b. What is happening at the arrow?

c. Explain what is the difference between the two constructs (i.e.,what is the interpretation of the graphs). 1 sentence.

d. Give the most likelybiological explanation for this difference.

e. Propose an experimentto test this explanation.

You are studying a cytosolic protein; your friend is studying a nuclear localization signal. How could you work together to show that his/her NLS is sufficient to cause any protein, including yours, to move to the nucleus. This will require several experiments (you must describe at least 3 experiments – think about showing what is specifically asked – think about control experiments). Please describe three experiments. So far, I have looked into protein localization using GFP and immunofluorescence but please expand on how these techniques could be used as an experiment and what a control experiment might look like for each experiment.

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