SOS help for GFP leakage during FACS - (Oct/06/2008 )
Hi
Is there anyone who may and could help me out. I am desperately disappointed.
I have to analyze the effect of an EGFP fusion protein on cell cycle. In the experiment I have to include the EGFP-N1 vector as control. My IF gives me good transfection efficiency. But during FACS my GFP signal is so poor. It seems that it is leaking because of using paraformaldehyde and NP-40 that I have to include in my PI staining solution. I have to permeabalize my cells to stain them with PI for CCA.
Is anyone using any kind of GFP which binds to membrane, or a vector having multiple copies of GFP? I do appreciate any kind of help and comments.
Thank you
-Haki-
QUOTE (Haki @ Oct 7 2008, 05:31 AM)
Hi
Is there anyone who may and could help me out. I am desperately disappointed.
I have to analyze the effect of an EGFP fusion protein on cell cycle. In the experiment I have to include the EGFP-N1 vector as control. My IF gives me good transfection efficiency. But during FACS my GFP signal is so poor. It seems that it is leaking because of using paraformaldehyde and NP-40 that I have to include in my PI staining solution. I have to permeabalize my cells to stain them with PI for CCA.
Is anyone using any kind of GFP which binds to membrane, or a vector having multiple copies of GFP? I do appreciate any kind of help and comments.
Thank you
Is there anyone who may and could help me out. I am desperately disappointed.
I have to analyze the effect of an EGFP fusion protein on cell cycle. In the experiment I have to include the EGFP-N1 vector as control. My IF gives me good transfection efficiency. But during FACS my GFP signal is so poor. It seems that it is leaking because of using paraformaldehyde and NP-40 that I have to include in my PI staining solution. I have to permeabalize my cells to stain them with PI for CCA.
Is anyone using any kind of GFP which binds to membrane, or a vector having multiple copies of GFP? I do appreciate any kind of help and comments.
Thank you
You could use a farnesylated-GFP, which binds to the membrane and cannot be released. Of course you should cotransfect it rather than clone your gene of interest in it because by changing the localization of your protein you could change its functions. You can easily find it in the literature and ask for it.
Good luck!
-dnafactory-
Hi dnafactory
Thank you for the info. I am looking for that vector.
Haki
QUOTE (dnafactory @ Oct 7 2008, 05:35 AM)
QUOTE (Haki @ Oct 7 2008, 05:31 AM)
Hi
Is there anyone who may and could help me out. I am desperately disappointed.
I have to analyze the effect of an EGFP fusion protein on cell cycle. In the experiment I have to include the EGFP-N1 vector as control. My IF gives me good transfection efficiency. But during FACS my GFP signal is so poor. It seems that it is leaking because of using paraformaldehyde and NP-40 that I have to include in my PI staining solution. I have to permeabalize my cells to stain them with PI for CCA.
Is anyone using any kind of GFP which binds to membrane, or a vector having multiple copies of GFP? I do appreciate any kind of help and comments.
Thank you
Is there anyone who may and could help me out. I am desperately disappointed.
I have to analyze the effect of an EGFP fusion protein on cell cycle. In the experiment I have to include the EGFP-N1 vector as control. My IF gives me good transfection efficiency. But during FACS my GFP signal is so poor. It seems that it is leaking because of using paraformaldehyde and NP-40 that I have to include in my PI staining solution. I have to permeabalize my cells to stain them with PI for CCA.
Is anyone using any kind of GFP which binds to membrane, or a vector having multiple copies of GFP? I do appreciate any kind of help and comments.
Thank you
You could use a farnesylated-GFP, which binds to the membrane and cannot be released. Of course you should cotransfect it rather than clone your gene of interest in it because by changing the localization of your protein you could change its functions. You can easily find it in the literature and ask for it.
Good luck!
-Haki-