Multi-spanning membrane protein - C terminal tagged (Nov/06/2008 )
Hello everyone,
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
-WHR-
QUOTE (WHR @ Nov 7 2008, 02:58 PM)
Hello everyone,
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
It should not be.
-Almasy-
QUOTE (WHR @ Nov 6 2008, 11:58 PM)
Hello everyone,
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
I very faintly suppose a different pH and of course a higher Ca2+ in ER lumen compared to cytosol which may effect GFP fluorescence;
to do it optimally, compare a C-terminal and N-terminal fused version...
-The Bearer-
QUOTE (The Bearer @ Nov 13 2008, 05:28 PM)
QUOTE (WHR @ Nov 6 2008, 11:58 PM)
Hello everyone,
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
A multi-spanning ER membrane protein was fused to GFP at its C-terminus. Dose the location(luminal or cytosolic) of GFP affect GFP fluorescent? Thank you.
I very faintly suppose a different pH and of course a higher Ca2+ in ER lumen compared to cytosol which may effect GFP fluorescence;
to do it optimally, compare a C-terminal and N-terminal fused version...
where is the membrane-spanning structure of your protein? in the C-terminus or N-terminus, in the middle??
I think you'd better not fuse GFP to the membrane-spanning structure.
Stone
-stone757-
Thank for your replies.
The protein of my interest is predicted to span the membrane 11 times. If the predicted topology is correct, the C-terminal tagged GFP will be luminal. The GFP fluorescence seems good.
-WHR-