Hello,
I've been trying to measure split YFP-constructs using a Tecan. The problem is, i can detect YFP as a whole construct without any problems (so the excitation/emission wavelengths are fine) but i cannot detect any signal when I combine my split YFP fragments (which i can detect under the microscope, so the complementation is happening.)
What I also have to mention is, that I'm measuring cell-free, so what i do is, I isolate the proteins and then measure them in the Tecan (same goes for whole YFP).
Did anyone experience similar problems?
Hi Sharanjah,
There can be different reasons for which you are experiencing issues with your measurements. May I ask if you are using the same concentrations of protein for both the whole and the complemented YFP? Differences in concentrations may require further adjustments of measurement parameters such as the "Gain" or the bandwidth.
Could you share some info about your settings, in particular which kind of Tecan reader you are using, the ex/em wavelengths, the bandwidth and the Gain for the measurements of the whole and of the complemented YFP?
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