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Increase Signal


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#1 fred_27

fred_27

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Posted 07 May 2009 - 12:35 AM

Hi,
I have a vector expressing two proteins (target protein and marker (EGFP)) separated by a sequence disturbing translation of the second protein (but that's what I want). Expression is driven by a CMV-promoter. Experiments are carried out in HEK293 cells.
My problem is, that the EGFP is expressed at very low levels (0.2%-2%) of the first protein and that it is not distingushable from background fluorescence in FACS or microscopy.
Now I would like to amplyfy the EGFP signal by a trick: instead of EGFP, the second protein should be a transcription factor that is able to induce a promoter that drives EGFP.
Is this possible? How much amplification can I expect? Which tf/promotor-combination(s) may work? Does anyone have vectors she/he may provide?

Best regards
Fred

#2 fred_27

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Posted 15 May 2009 - 01:36 AM

Hi,
as many people have read this question, I want to share the informations I've found.
There is the UAS/Gal4-System used in drosophila and for mammalian yeast 2 hybrid (Clontech, Matchmaker).
I'm not sure if there are other Promotor/Transcriptionfactor-Systems. If anybody knows an alternative system I'm interested.

As I only want to check whether my idea will work (enhancing a signal from 0.2%-2% to a detectable EGFP expression) and we are a very small group, I can not afford buying e.g. the Matchmaker for Mammalian Y2H just for fun (or just to find out).
Has anybody UAS/Gal4 vectors usable in e.g. HEK293 cells and is willing to send me some DNA on a filter paper?


Thanks in advance
Fred




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