1) You have identified a channel that is involved in pumping of Fe2+ ions into t
ID: 96025 • Letter: 1
Question
1) You have identified a channel that is involved in pumping of Fe2+ ions into the liver cell. The channel appears to be regulated by a gating mechanism. You wish to verify that conformation change involving the gating is involved in closing the channel when excess Fe2+ ions enter the cells. Which of the following methods would you choose for your experiment?
2) Cathy is a student in a lab studying the role of a transporter 'X' in response to Adrenaline hormone. Her initial studies have revealed that the activity of the receptor is increased in response to adrenaline. She is seeking your advice to plan an experiment to look at localization of the receptor and tocheck if presence of adrenaline leads to increased accumulation of receptor 'X' to the membrane. Choose the best experimental strategy on her behalf.
a. Purify the proteins from the cells and perform a western blot with a Mouse antibody against transporter 'X'.
A. Tag the gating domain with GFP and use FRAP to show that gating is required for the closure of the channel.Explanation / Answer
Answer 1:- GFP is a Green fluorescent Protein which when exposed to light in the blue to ultraviolet range shows bright green fluorescence. The tagging of GFP is done by the method of Photobleaching and after that the technique known as FRAP(Fluoroscence Recovery After Photobleaching) it is a technique to determine the diffusion mainly in living cells and it uses fluoroscence microscopy. The easiest way to determine the involving of the gating mechanism is this method.
FRET analysis requires use of chromophores because of the light emission and the transfer of energies from one chromophore to the another. The tagging of the gate and the channel with appropraite fluorescent protein does not make it a chromophore, hence, the FRET analysis does not work with this type of method and the determination of the gating mechanism is quite impossible by this method.
So the Option B is incorrect.
As mutations are one of tthe many natural processes that occur in nature. The mutation by any artificial mechanism lacks accuracy and preciseness. The artificial mutation is quite tough job to do and has many abnormalities which sometimes result into cell apoptosis.
Option C and D arre related to mutation and both the optopns has the same reason. Hence, the Option C and D are incorrect.
The most accurate and precise is Option A.
Answer :- Our main task is to prove taht the receptors at the cell membrane increases in the presence of Adrenaline and purifiaction of protein and interaction with the mouse antibody does not reveal the increase in the amount of receptors because the process only occurs inside the cell.
So the Option A is incorrect.
FRET(Fluorescence Resonance Energy Transfer), it is a interaction between two chromophores or fluorophores. When we use FRET, a fluorophore instead of emitting light it transfers energy to other or acceptor fluorophore which is at minimum distance and then the acceptor fluorophore emits visible light ,this occurs when it get exposed to the light source. In our experiment CFP and GFP both are fluorophores and when tagged with adrenaline and transporter X, in FRET assay the fluorophores are tagged and exposed. The minimum distance is maintained because of the adrenaline and receptor interaction, which would show the increase in number of the receptor.
So the Option B is correct.
When only the Transporter X is tagged with the protein is hard to determine the increase in number of the receptor in the presence or absence of adrenaline is happening or not. The reason behing is this that it may be possible that when the adrenaline is secreted some other hormone or protein might be also secreted with adrenaline due to which the number of receptors is increased.
So the Option C is incorrect.
As the researcher has only revealed that there is a change in the number of Transporter X in the presence of adrenaline. There is still no report about the presence of ATP during the whole mechanism. The method would be totally hit and trial without any single assurance of the results.
So the Option D is incorrect.
Related Questions
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.