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Luciferase Assay Weird Result: TK-Renilla Changing More than SV40 Firefly

luciferase renilla firefly

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#1 pinhead

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Posted 17 May 2012 - 04:11 AM

Hi Everybody,

I'm getting strange results in Luciferase assay, Im wondering if anyone has had a similar experience.

I'm quite experienced in luciferase assays. We're trying to study gene activator proteins using a standard setup:
Cotransfecting HEK cells in 24well plates with (1) UAS-SV40-Firefly [FF] 100ng, (2) Gal4-Activator fusion in pcDNA3.1 800ng, (3) TK-Renilla[RL] 20ng.

Transfection is fine, luciferase signal is fine, replicates are tight. The amount of transfected DNA is kept constant in all experiments, by adding empty expression plasmid to the mix where necessary.

I EXPECT to see specific activation of the FF construct with the cotransfected fusion protein. When I do the experiment and normalise FF/RL, I get a very nice effect: addition of the fusion activator increases the value of FF/RL in comparison to various different controls and irrelevant proteins that show no difference from background FF/RL value.

HOWEVER, when I look closely at the data, I see that the effect on FF/RL in fact comes from DECREASE in Renilla signal, rather than increase in Firefly. This is consistent between experiments. In fact, if you look at the FF levels alone, you would observe no effect.

However, the FF/RL behaviour is consistent with the effect occurring through the FF plasmid, since all the controls I try (eg omit UAS sites from FF plasmid or expressing Activator with incorrect DNA binding domain etc) show no increase in FF/RL compared to background.

Any ideas about this? Anybody with similar experiences?

Your help is much appreciated!
Rory

#2 T.G.S

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Posted 10 June 2012 - 05:19 PM

Hello Guys

Hi dude if you figure out this problem please share with us. I am too faced with similar problem, renilla signal jumps wherever it wants. I am sure it is not due to transfection efficiency, (condition is 10ng renilla reporter+200ng firefly reporter and 200ng GFP+ balancing empty vector up to 500ng by PEI method) I prepared this mixture in one tube in 600ul for 3 well ( total 500ng DNA x 3=1500ng) then added into 3 wells dividing by 200.
But result is 1. firefly 3308752 renilla 1920394
2. firefly 6475843 renilla 194085
3. firefly 5841767 renilla 1587421
Guys help me to figure out





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