Protein targetting - (Jun/02/2009 )
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
-MaggieRoara-
MaggieRoara on Jun 2 2009, 09:47 PM said:
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
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
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.
-Dr Teeth-