is my GFP quenched? - (Apr/02/2008 )
hello everybody
I have selected stable cell lines transfected with VHH-GFP ( camel antibody-GFP ), RT-PCR result shows that the selected cells express both VHH and GFP, but I can't see any fluorescence under microscope.
could anybody give any hints?
tnx
-yasharsadian-
Are the antibody and GFP connected as proteins or just expressed from the same promotor (with an IRES or so)?
Maybe (if connected) gfp can't fold correctly? What GFP are you using, they differ quite a lot in properties (wild type exciation is optimal around 400nm wheras enhanced gfp has a max around 480)?
-vairus-
QUOTE (vairus @ Apr 2 2008, 10:13 PM)
Are the antibody and GFP connected as proteins or just expressed from the same promotor (with an IRES or so)?
Maybe (if connected) gfp can't fold correctly? What GFP are you using, they differ quite a lot in properties (wild type exciation is optimal around 400nm wheras enhanced gfp has a max around 480)?
Maybe (if connected) gfp can't fold correctly? What GFP are you using, they differ quite a lot in properties (wild type exciation is optimal around 400nm wheras enhanced gfp has a max around 480)?
tnx for replying, they are connected by prolin linker
I use EGFP.
until 2 weeks after transfection I can see GFP but since then it becomes weak and dispersed.
-yasharsadian-