Expression of GFP and DsRed tagged proteins - co-localization needed for FRET (Jul/07/2008 )
I am trying to do FRET invivo by acceptor photobleaching. Since I am not able to have co-localization, FRET is impossible, however these WT proteins are known to interact in-vivo, so they should show high FRET effeciency . My overall aim is to use different truncated mutants of 1 protein to determine the protein-protein interaction site with the 2nd one.
My constructs are fusion of GFP and DsRed, my question is why am i seeing expression of GFP tagged fusion protein mostly in nucleus than cytoplam where proteins are mostly synthesized?
DsRed tagged proteins are expressing in cytoplasm.
Any comment/ suggestion/advise is appreciated.
Thanks !
My constructs are fusion of GFP and DsRed, my question is why am i seeing expression of GFP tagged fusion protein mostly in nucleus than cytoplam where proteins are mostly synthesized?
DsRed tagged proteins are expressing in cytoplasm.
Any comment/ suggestion/advise is appreciated.
Thanks !
Perhaps your GFP-tagged protein is localized mainly in the nucleus.
Beware: I have heard DsRed tag can dimerize by itself! Although this is just hearsay, if somebody knows it for sure, jump in!
My constructs are fusion of GFP and DsRed, my question is why am i seeing expression of GFP tagged fusion protein mostly in nucleus than cytoplam where proteins are mostly synthesized?
DsRed tagged proteins are expressing in cytoplasm.
Any comment/ suggestion/advise is appreciated.
Thanks !
Perhaps your GFP-tagged protein is localized mainly in the nucleus.
Beware: I have heard DsRed tag can dimerize by itself! Although this is just hearsay, if somebody knows it for sure, jump in!
Can we use a different tag to make it express in cytoplasm ?
what can be the possible reason, does my GFP fusion protein express in cytoplam and moves to nucleus??
i am using HeLa cells, is changing cell line make a difference in expression?
Thanks !
Yes you can, if you make a fusion protein. But what is the point? You need to see the interaction where your protein naturally resides.
what can be the possible reason, does my GFP fusion protein express in cytoplam and moves to nucleus??
Yes. Your protein should have NLS.
i am using HeLa cells, is changing cell line make a difference in expression?
I doubt it.
Thanks !
Yes you can, if you make a fusion protein. But what is the point? You need to see the interaction where your protein naturally resides.
since these WT proteins are know to interact thoroughtout the life span, just because they are not expressing in the same location in HeLa cells,,,,,,we can not do FRET and that means we can not determine the interacting regions, so if i can make them co-express, i might be able to narrow down the region of interaction using my mutants
what can be the possible reason, does my GFP fusion protein express in cytoplam and moves to nucleus??
Yes. Your protein should have NLS.
what is NLS?
Thanks !
i am using HeLa cells, is changing cell line make a difference in expression?
I doubt it.
Thanks !
Yes you can, if you make a fusion protein. But what is the point? You need to see the interaction where your protein naturally resides.
since these WT proteins are know to interact thoroughtout the life span, just because they are not expressing in the same location in HeLa cells,,,,,,we can not do FRET and that means we can not determine the interacting regions, so if i can make them co-express, i might be able to narrow down the region of interaction using my mutants
You want to check the interaction in their native environment. So use the cells where your protein is endogenously expressed. You would prove nothing if the mutants don't interact in HeLa cells. Waste of time, in my view.
what can be the possible reason, does my GFP fusion protein express in cytoplam and moves to nucleus??
Yes. Your protein should have NLS.
what is NLS?
Thanks !
Nuclear localization signal
i am using HeLa cells, is changing cell line make a difference in expression?
I doubt it.
Thanks !
You are most welcome! If you don't mind my advising, I would suggest more reading on your part.
Yes you can, if you make a fusion protein. But what is the point? You need to see the interaction where your protein naturally resides.
since these WT proteins are know to interact thoroughtout the life span, just because they are not expressing in the same location in HeLa cells,,,,,,we can not do FRET and that means we can not determine the interacting regions, so if i can make them co-express, i might be able to narrow down the region of interaction using my mutants
You want to check the interaction in their native environment. So use the cells where your protein is endogenously expressed. You would prove nothing if the mutants don't interact in HeLa cells. Waste of time, in my view.
what can be the possible reason, does my GFP fusion protein express in cytoplam and moves to nucleus??
Yes. Your protein should have NLS.
what is NLS?
Thanks !
Nuclear localization signal
i am using HeLa cells, is changing cell line make a difference in expression?
I doubt it.
Thanks !
You are most welcome! If you don't mind my advising, I would suggest more reading on your part.
Absolutely not , if you have some particular reference or links, I would be happy to read them as well
Thanks!
If your overall aim is to determine the interaction site between these two proteins, I am very confused as to why you would want to use FRET. FRET is a very intricate, difficult assay. Have you tried FRET with the tags on both the amino-terminus (NT) and carboxy-terminus (CT)? You may not be getting the signal because the tag on one (or both) protein is on the wrong side. If the interaction occurs on the CT and your tag is on the NT, you may not get the signal even though the proteins are interacting. It seems to me that GST pulldowns or Co-IP from lysates of expressing cells is a much better way to go. You can make all sorts of truncation and mutants and define the region of the proteins necessary for the interaction much easier and faster. I agree with cellcounter that once you start making your protein be localized artificially, you really have lost the entire point of the experiment. Where are the native, endogenous proteins localized? Are they regulated by cell cycle? Under what conditions have these two proteins been identified as interacting (for example, were the cells synchronized)? You need to check to see if the localization of your expressed proteins matches that of the endogenous proteins. Otherwise you might be seeing a non-specific affect of the tag which could also be changing the binding patterns of the protein. A large tag such a GFP or dsRed can mask domains and signals normally exposed in the protein causing changes in localization, binding, activity and stability.
Thanks!
'm glad
http://search.vadlo.com/b/q?sn=158621799&a...=FRET&rel=2
http://search.vadlo.com/b/q?sn=158621799&a...al%22&rel=2
What RK says here is absolutely correct...
There really are much simpler assays. Fret is difficult even with proteins in which the interaction is well understood. Can you answer the questions RK asked?