quenching of GFP after selection of stable cells - (Apr/02/2008 )
hello
I transfected my CHO cells with antibody-GFP and started selection with hygro, after 30 days of selection, I amplified resistant cells and on the 60th day I had RT-PCR on both genes and result were + for both genes it is noteworthy that they are connected by prolin linker.
at early days after transfection I can fluorescence but as intense as non-fused GFP, but at the 60th day I dont see fluorescence although I could prove existance of GFP mRNA.
what happens to my GFP?
I do not have anti-GFP to detect on western blot, therefore what should I do?
-yasharsadian-
QUOTE (yasharsadian @ Apr 2 2008, 12:05 PM)
hello
I transfected my CHO cells with antibody-GFP and started selection with hygro, after 30 days of selection, I amplified resistant cells and on the 60th day I had RT-PCR on both genes and result were + for both genes it is noteworthy that they are connected by prolin linker.
at early days after transfection I can fluorescence but as intense as non-fused GFP, but at the 60th day I dont see fluorescence although I could prove existance of GFP mRNA.
what happens to my GFP?
I do not have anti-GFP to detect on western blot, therefore what should I do?
I transfected my CHO cells with antibody-GFP and started selection with hygro, after 30 days of selection, I amplified resistant cells and on the 60th day I had RT-PCR on both genes and result were + for both genes it is noteworthy that they are connected by prolin linker.
at early days after transfection I can fluorescence but as intense as non-fused GFP, but at the 60th day I dont see fluorescence although I could prove existance of GFP mRNA.
what happens to my GFP?
I do not have anti-GFP to detect on western blot, therefore what should I do?
a mRNA signal in pcr does not necessarily mean protein expression, however, there will be some chance to find protein expression; the prediction from pcr result how much protein is really expressed is uncertain and should be shown by a specific antibody;
may be the constitutive protein expression is too low; try to use an inducible promoter;
or it is a technical problem; normal fluorescent light is often to harsh even for EGFP; better use confocal microscopic imaging
-The Bearer-