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Transfection Forum Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


GFP detection

GFP detection - Transfection Forum

GFP detection - Transfection Forum. Cell transfection discussion including stable cells transfection, transient transfection. Troubleshooting and posting of discussions on lipofection, and reagents: Fugene, lipofectamine, and others.


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  #1  
Old 07-09-2010, 07:35 PM
Pipette Filler
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Default GFP detection



Hi,
I have a cell line that is stably expressing Proteinx-GFP. After some time, there are cells, where I cannot detect GFP under the microscope . My question: is that only the case, if proteinX isn`t expressed anymore or is it possible, that it is still expressed, but GFP doesn`t show fluorescence anymore?
Thank you!
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Old 07-13-2010, 04:37 PM
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Default Re: GFP detection

I can't answer this absoluty, because both of your ideas are possible. You should ask which case appears under what condition. First of all it is important if your GFP Signal reduces within time of living of one passage of the cells or does it appear as cause of many transfections. In first case your cells are possibly dead (are you using suspension or adherence cells ?). But I suggest you mean the effect appears after some passaging of your cells. So in this case there must be considered that (a bit dependend on the cell line you use) every passage (the process itself: change medium (fluid movement), transport flask, control under microscope) is a physical stress to the cells and so they will react on it. A mutation (just about two or three amino acids are enough) can cause loss of absorption at a specified wavelenght (For instance mutation at Ser65 and Glu222 in wild type GFP cause loss of absorption at 400nm). Loss of N-terminal Methionin or truncation of more than 7 bases at the C-terminus also kills the signal. Inhibited transcription through ribo switches or posttranslational modification may also be possible. Even important is if the GFP expression is triggered through an external component. Furthermore there are differnt chemical substances that can reduce GFP activity. For instance some oxyradicals (-> cell stress ?) or chlorine solutions (pH dependend). By the way when you write you have a cell line stably expressing Protein-GFP do you mean the cell line was stable transfected or just your GFP signal is continiously produced ?? And when you write Protein-GFP you mean the cell is expressing the Protein called GFP or do you mean its expressing a Protein-GFP complex (then: how this complex is generated?, is the GFP signal dependend to the protein of the complex, could this protein be destroyed causing signal loss through any mechanism ?). Im not a big expert, but I hope this helped a little bit finding the reason for your problem.
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Old 07-20-2010, 10:38 PM
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Default Re: GFP detection

Hi Omicron,
thank you very much for your help! My protein of interest is fused to GFP and should be constantly expressed. The cells were stably transfected and I loose the signal over generations. Maybe also within the life of a single cell, but I can`t follow that. Sounds strange
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Old 07-26-2010, 07:50 PM
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Default Re: GFP detection

Hmm, when you loose the signal over generations and your GFP is a fusion protein with the one of your interest you can check the presence of the protein of interest. Then you can see if just the GFP signal is lost or if the protein complex is not expressed anymore. Do an ELISA of the protein of interest or RT-PCR. You can also do an additional normal PCR to the GFP sequence. Then you know if the cells are really stable transfected, or if they are not and thats simply the cause of signal loss.
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Old 12-11-2010, 09:38 PM
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Default Re: GFP detection

If your protein is toxic to your cells you might select cells with less expression over time. I had a good experience with stable lines and inducible promoters for toxic proteins.
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Old 02-17-2011, 06:35 AM
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Default Re: GFP detection

Hi
I am actually having the same problem with my stable cell line (GFP). I was wondering if you found out what the problem was?
thanks
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Old 02-17-2011, 11:13 AM
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Default Re: GFP detection

have you checked your cloning of the insert? is it in frame? no mutations?
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Old 02-17-2011, 11:50 AM
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Default Re: GFP detection

I didnt make the stable cells line. I got it from a different lab, and it seems I am the only one having this problem! i ahve GFP expression for a few passages and then for no apparent reason it just stops!
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Old 02-18-2011, 02:33 PM
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Default Re: GFP detection

It depends on the protein you are overexpressing, it is toxic you will loose the expression over time, one of the reasons for inducible promoters.
Are you growing your cells in medium which is supplemented with the appropriate antibiotic to maintain selection pressure?
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