Hi everyone,
I get no luciferase or renilla luciferase signal from my samples.
Briefly, I agro-infiltrated tobacco leaves with a positive control construct (CsMV-bZIP60-nosT -- promBiP3-firefly.luciferase-nosT -- CsMV-renilla.luciferase-nosT) prior to introducing my test constructs. Using this construct, I should get a signal from both firefly and renilla luciferase. I get values that range from 199 - 380 for both, with similar values for infiltrated and non-infiltrated leaves.
I thought it was an issue with transfection, however, I ran a protein extract on a gel, blotted it and probed for renilla luciferase. I see renilla luciferase (see image), so it is expressed. However, the size of the protein on the blot appears to be smaller than 36kDa. The prominent band around 25kDa is absent from the negative control though.
Then I thought that it might be an issue with the samples being too concentrated. I did various dilutions, and still, no signal. I am hoping someone else has experience with doing this assay from plant tissues. Please, if you have any advice, please shoot!!! Much appreciated.
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