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CFP YFP - relocation (Oct/03/2007 )

HI

I did a fused protein with YFP and CFP and when I check on the microscop I see the fluorescence only in the cytoplasm.
this protein should be everywhere.

Is it know that CFP or YFP tag can make the protein staying in the cytoplasm.

thanks

-ulujm-

What is it that makes this protein able to localize to the nucleus? Does it have it's own NLS or does it require an interaction? Could be because the large tag is covering up the NLS or disrupting a critical interaction. What about your fluorescence control (ie: CFP and YFP alone)? I've worked with RFP and never had an issue with localization. You may need to move the tag to the other terminus. Also, some proteins, such as yeast MCM5, simply won't take a terminus tag (CT or NT) and still be functional.

-rkay447-

QUOTE (rkay447 @ Oct 3 2007, 02:29 PM)
What is it that makes this protein able to localize to the nucleus? Does it have it's own NLS or does it require an interaction? Could be because the large tag is covering up the NLS or disrupting a critical interaction. What about your fluorescence control (ie: CFP and YFP alone)? I've worked with RFP and never had an issue with localization. You may need to move the tag to the other terminus. Also, some proteins, such as yeast MCM5, simply won't take a terminus tag (CT or NT) and still be functional.



the protein is know to be everywhere cyto and nuc. shown by immunostaining.

yes I have a controle of FP or YFP alone and fluo is everywhere.

yes I may try in other orientation

-ulujm-

QUOTE (ulujm @ Oct 3 2007, 02:03 PM)
HI

I did a fused protein with YFP and CFP and when I check on the microscop I see the fluorescence only in the cytoplasm.
this protein should be everywhere.

Is it know that CFP or YFP tag can make the protein staying in the cytoplasm.

thanks


may be the two tags make the protein too large to enter the nucleus

-The Bearer-