Destroying GFP fluorescence - (Sep/19/2007 )
I've got a rather strange question. I am doing immunofluorescence on cultured cells and have a GFP-tagged construct. I would like to actually destroy the GFP signal so I can stain another protein in the green channel. I've tried photobleaching and ethanol incubations to no avail. Otherwise, I know a strong acid will denature GFP but it also destroys my other desired epitopes. I've also read that freezing tissue specimens will destroy GFP but I'm worried that this will mess with my cellular morphology. Does anyone have any other ideas?
-rkay447-
QUOTE (rkay447 @ Sep 19 2007, 03:38 PM)
I've got a rather strange question. I am doing immunofluorescence on cultured cells and have a GFP-tagged construct. I would like to actually destroy the GFP signal so I can stain another protein in the green channel. I've tried photobleaching and ethanol incubations to no avail. Otherwise, I know a strong acid will denature GFP but it also destroys my other desired epitopes. I've also read that freezing tissue specimens will destroy GFP but I'm worried that this will mess with my cellular morphology. Does anyone have any other ideas?
we use our UV-illuminator to destroy GFP; why your photobleaching does not work?
-The Bearer-
QUOTE (The Bearer @ Sep 20 2007, 05:54 AM)
QUOTE (rkay447 @ Sep 19 2007, 03:38 PM)
I've got a rather strange question. I am doing immunofluorescence on cultured cells and have a GFP-tagged construct. I would like to actually destroy the GFP signal so I can stain another protein in the green channel. I've tried photobleaching and ethanol incubations to no avail. Otherwise, I know a strong acid will denature GFP but it also destroys my other desired epitopes. I've also read that freezing tissue specimens will destroy GFP but I'm worried that this will mess with my cellular morphology. Does anyone have any other ideas?
we use our UV-illuminator to destroy GFP; why your photobleaching does not work?
I was trying to photobleach with a strong white light which brought the signal down but not completely. I'll try our DNA imager. Which wavelength did you use? We have the capability of 302 or 365. Also, how long did you need to expose your cells to UV before complete loss of GFP signal? Thanks for the idea.
-rkay447-
Why bother? There are so many dyes to chose from.
-genehunter-1-
QUOTE (rkay447 @ Sep 21 2007, 10:32 AM)
QUOTE (The Bearer @ Sep 20 2007, 05:54 AM)
QUOTE (rkay447 @ Sep 19 2007, 03:38 PM)
I've got a rather strange question. I am doing immunofluorescence on cultured cells and have a GFP-tagged construct. I would like to actually destroy the GFP signal so I can stain another protein in the green channel. I've tried photobleaching and ethanol incubations to no avail. Otherwise, I know a strong acid will denature GFP but it also destroys my other desired epitopes. I've also read that freezing tissue specimens will destroy GFP but I'm worried that this will mess with my cellular morphology. Does anyone have any other ideas?
we use our UV-illuminator to destroy GFP; why your photobleaching does not work?
I was trying to photobleach with a strong white light which brought the signal down but not completely. I'll try our DNA imager. Which wavelength did you use? We have the capability of 302 or 365. Also, how long did you need to expose your cells to UV before complete loss of GFP signal? Thanks for the idea.
it depends on kind of GFP (original, green latern, EGFP etc) which differ in stability towards light; we have a 254 nm-illuminator which work by strong excess; you may optimize the conditions
-The Bearer-
QUOTE (genehunter-1 @ Sep 21 2007, 03:14 PM)
Why bother? There are so many dyes to chose from.
I'm using the GFP to look at localization of an expressed protein. I can analyze my cells live by confocal microscopy for this localization and then I want to fix the cells and use immunofluorescence to look at other proteins. Problem is we only have three filters and I need all three for other desired stains so I have to be able to get rid of the GFP signal. I thought this would be an easy task but is turning out to be the most difficult.
-rkay447-