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Protein targetting


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#1 MaggieRoara

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Posted 02 June 2009 - 05:47 PM

Hi everyone!

I have the cytoplasmic domain of a membrane protein that i tagged with myc and expressed in HEK293 cells. This protein is thought to be a synaptic protein. I ran the protein sequence througha nuclear localisation signal prediction software and there is reason to believe that a large portion of my cytoplasmic domain is a nuclear localisation signal. However, when i do immunoflorescence, the protein is in the ER. I did a z-sectioning of the cells as well, and I have reson to believe that the protein is present in both the nucleus and the ER. Can someone please help me with this. I am not very familiar with protein targetting.


Thank you

:D

#2 Dr Teeth

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Posted 04 June 2009 - 04:36 AM

Hi everyone!

I have the cytoplasmic domain of a membrane protein that i tagged with myc and expressed in HEK293 cells. This protein is thought to be a synaptic protein. I ran the protein sequence througha nuclear localisation signal prediction software and there is reason to believe that a large portion of my cytoplasmic domain is a nuclear localisation signal. However, when i do immunoflorescence, the protein is in the ER. I did a z-sectioning of the cells as well, and I have reson to believe that the protein is present in both the nucleus and the ER. Can someone please help me with this. I am not very familiar with protein targetting.


Thank you

:huh:


Many membrane proteins are synthesized in the ER, so it could be that the functional protein is located in the nucleus while the bulk of the protein while being synthesized is at the ER, yielding visible protein at both locations. If the protein is an ER resident protein, it should have an ER retention signal (usually KDEL). Since nuclear localization signals are quite variable, the best way to test is to mutate some of the basic residues of the NLS and see if the protein fails to undergo nuclear localization.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley




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