7 replies to this topic

[AllenChiu]

My GFP fused protein have a large proportion of mislocalization when overexpressed by transfection.
It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.
Do you have any experience like this?Do you have any suggestion?
Thanks very much.

[Curtis]

AllenChiu, on Apr 10 2010, 06:39 AM, said:

It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.

so is it localized to nucleus or not? I don't understand

[AllenChiu]

Curtis, on Apr 10 2010, 11:35 PM, said:

AllenChiu, on Apr 10 2010, 06:39 AM, said:

It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.

so is it localized to nucleus or not? I don't understand

well，can't you see the difference between nucleus and nucleolus?

[Curtis]

AllenChiu, on Apr 10 2010, 08:20 AM, said:

Curtis, on Apr 10 2010, 11:35 PM, said:

AllenChiu, on Apr 10 2010, 06:39 AM, said:

It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.

so is it localized to nucleus or not? I don't understand

well，can't you see the difference between nucleus and nucleolus?

oops sorry, wasn't wearing my glasses....)) seriously

[AllenChiu]

Curtis, on Apr 11 2010, 08:50 AM, said:

AllenChiu, on Apr 10 2010, 08:20 AM, said:

Curtis, on Apr 10 2010, 11:35 PM, said:

AllenChiu, on Apr 10 2010, 06:39 AM, said:

It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.

so is it localized to nucleus or not? I don't understand

well，can't you see the difference between nucleus and nucleolus?

oops sorry, wasn't wearing my glasses....)) seriously

Never mind：）

[bob1]

AllenChiu, on Apr 10 2010, 06:39 AM, said:

My GFP fused protein have a large proportion of mislocalization when overexpressed by transfection.
It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.
Do you have any experience like this?Do you have any suggestion?
Thanks very much.

My suggestion is not to use GFP as a tag.  The reasons for this are: 1) it is a gigantic tag, so it may be affecting protein synthesis. 2) you are running it off a plasmid, massive over-production of a tag of this size could be causing toxic/stress effects.  3) Big tags may be affecting active portions of the tagged protein through steric or physical hindrance. 4) It's not localising correctly, therefore it is probably not functioning properly.

[AllenChiu]

bob1, on Apr 12 2010, 08:23 AM, said:

AllenChiu, on Apr 10 2010, 06:39 AM, said:

My GFP fused protein have a large proportion of mislocalization when overexpressed by transfection.
It is supposed to localize in nucleolus,but actually in 90% of the GFP positive cell it localized in nucleus.
Do you have any experience like this?Do you have any suggestion?
Thanks very much.

My suggestion is not to use GFP as a tag.  The reasons for this are: 1) it is a gigantic tag, so it may be affecting protein synthesis. 2) you are running it off a plasmid, massive over-production of a tag of this size could be causing toxic/stress effects.  3) Big tags may be affecting active portions of the tagged protein through steric or physical hindrance. 4) It's not localising correctly, therefore it is probably not functioning properly.

I also highly suspected massive over-production of GFP tag could be causing toxic/stress effects,do you know any paper that had investigated this seriously?
By the way,my interested protein is a very weird protein,which can only be detected when fused to GFP tag instead of Flag or Fc tag.Have you heard of anything like this?

[bob1]

I guess it is possible that all the antibodies against your protein are against denatured forms.  Try looking for ones that are for the native protein (validated for IF or IHC).  I would be surprised if there are no other tags that work for the protein, though I am using an HA tagged protein at the moment,  that seems have some steric hindrance for detection with an anti-HA antibody using an N-terminal tag, compared to a C-terminal tag.