Protein in the nucleus vs in the cytoplasm

[yi198720022004]

Hello,
If I have a gene X that is regulated by controlling how much of the protein is in the nucleus versus in the cytoplasm. Can anybody tell me what kind of experiment you will perform if you want to find out if gene X is indeed regulating the protein level. What type of experiment will allow me to distinguish the protein level in the nucleus vs in they cytoplasm? Thank you for your help

[JackBean]

mutate the gene X to either silence or constitutively express and watch the localisation of your protein. Or just induce in some way

[jwalin]

Hello,
If I have a gene X that is regulated by controlling how much of the protein is in the nucleus versus in the cytoplasm. Can anybody tell me what kind of experiment you will perform if you want to find out if gene X is indeed regulating the protein level. What type of experiment will allow me to distinguish the protein level in the nucleus vs in they cytoplasm? Thank you for your help

I aint sure if i completely understand your question. could you be a little clearer?

[yi198720022004]

How can you watch the location of the protein??

[canalon]

gfpor other fluorescent protein fusion would be one possibility (if it does not interfere with function). Otherwise fixation and use of antibody a posteriori

[jwalin]

@yi98...
I sent you a PM but it stayed in my outbox so am posting it here
The following was your message to me:

I will try to explain more clearly this time. That's say if I found gene X that may function as an inhibitor of the NF-kB pathway. I want to design an experiment to show that gene X does indeed target the NF-kB pathway.
I learn that NF-KB is primarily regulated by controlling how much of the protein is in the nucleus versus the cytoplasm. So I want to design a experiment that allows me to measure the of protein in the nucleus vs in the cytoplasm to prove that gene X indeed target NF-kB pathway. Could you please help me? I was thinking do a nucleus prep but I was not sure how. Thank you

Okay so I think i get your point now. Though one clarification before i can answer your question. Over here you discussed two things that control nfkb pathway,
1. the relative amount of proteins and
2. gene X.
So basically you want to know the relative amount so that you can control it or study systems in which it remains above a certain threshold and as a result you can determine if the gene X affects the nfkb pathway or not.

A) Okay if that is the case then i have one question the nf-kb pathway is regulated by the net amount of proteins or by the amount of some particular protein?
i) If its the net amount then it will be a little hard. hmm let us put that on hold and not discuss that now.
ii) But if its the amount of some particular protein then what you can do is add GFP (green fluoroscent protein) coding gene fragment to the gene for the protein and check and compare the relative fluoroscence. There are other methods too but in all of them you are basically tagging the protien with something that something can be seen like fluoroscence or perhaps radioactivity....

B) If i haven't understood you correctly please explain. I exactly dont know what does the nfkb pathway do if it controls the relative amount of protein then the whole experiment becomes a lot easier. to check if the gene X regulates nfkb pathway you just need to measure the relative amount of proteins and that can be done as I discussed above.


Hope that helps.