Question

1- Why is the FRAP technique applicable only to live cells?

2- A researcher wishes to confirm that the adenine nucleotide translocase (ANT) is localized to mitochondria by using immunofluorescence microscopy. He has an anti-ANT antibody fluorescently labelled with Alexa Fluor red and plans to show co-localisation with mitochondria using mitotracker red. Why will this not work? Suggest a better approach.

3- Briefly describe the principle of the FISH technique and indicate one type of sample that this technique could be used with.

4- You are working in a lab that had access to a range of deletion collections. A company approaches your supervisor with a new drug that has great anti-tumor properties and also inhibits the growth of yeast. The company wants to check that the mechanism of action of the drug is novel. That is, it is not similar to other known anti-tumour compounds. what techniques could you use to:

a. Demonstrate the drug has a different mechanism of action from known anti-tumor compounds.

b. Elucidate the mechanism of action of the drug

Answer 1

1- Why is the FRAP technique applicable only to live cells?

The flourescence recovery after photobleaching is one of the techniques in which there would be determination of diffusion kinetics. There oils be quantification of 2d diffusion within thin molecular films that would be containing probes that are flourescently labeled so that there would be examination of single cells. This is an useful technique in studying the diffusion properties of the biological membranes as well as the properties of protein binding. Also, there would be characterisation of the free energy of surface structures along with their hydrophilic characterisation.

In order to determine diffusion kinetics, living cells would be required. Unless a cell is alive, there will not be movement of molecules, and thus diffusion kinetics cannot be studied. Also, unless a cell is live, it will not be fluorescing. Also, unless there is active molecule movement in the cells, there will not be replacement of the bleached fluorophores with the molecules that are unbleached. Hence, live cells would be required.