I made GFP-Alexa dye bioprobes and tested them in HeLa cells. Then, in both in vitro and in vivo conditions, I assessed FRET efficiency. In certain cases, in vivo results are superior to those obtained in vitro. As a result, I'm perplexed.
What do you mean by "in vivo results are superior to those obtained in vitro"? Do you mean that the FRET efficiency is greater in vivo than in vitro? How have you measured this?
Adam B Shapiro Thank you very much for your cordial response. Yes, FRET efficiency is greater in vivo than in vitro. I have measured FRET efficiency by following equation = Acceptor emission (Alexa dye) / Donor emission (GFP)
I'm trying to understand the experiment. Did you prepare the Alexa-labeled GFP by chemical labeling of GFP in vitro? Did you inject it into HeLa cells or allow the HeLa cells to take it up from the medium by pinocytosis? What is the bioprobe intended to measure, degradation of the GFP or something else? Is the protein in the cytoplasm or in the lysosome-endosome compartment?
Do you see that the GFP emission is lower in vivo than in vitro, that the Alexa emission is higher, or both?
On the following, I will provide you with the answers to your queries.
Q: Did you prepare the Alexa-labeled GFP by chemical labeling of GFP in vitro?
Ans: I have prepared Alexa -GFP labeled bioprobe and checked enzyme activity (caspase-3) in vitro. In this case, donor emission increases (GFP) because enzyme cuts the amino acid sequences; as a result FRET efficiency lower than the normal bioprobe.
Q: Did you inject it into HeLa cells or allow the HeLa cells to take it up from the medium by pinocytosis?
Ans: No, endocytosis
Q: What is the bioprobe intended to measure, degradation of the GFP or something else?
Ans: Bioprobe digestion by caspase-3 and measured FRET efficiency.
Q: Is the protein in the cytoplasm or in the lysosome-endosome compartment?
Ans: cytoplasm and nucleus (sensitivity to caspase-3)
Q: Do you see that the GFP emission is lower in vivo than in vitro, that the Alexa emission is higher, or both?
Ans: I utilize a fluorescence spectrophotometer in vitro and then determine FRET efficiency. In vivo, however, I performed flow cytometric tests and calculated FRET efficiency after introducing the bioprobe into the cell.
In HeLa cells, in vivo findings are sometimes higher than in vitro results (FRET efficiency), while in vitro results are sometimes higher than in vivo. Then I'm worried.
One concern I have is that the GFP bioprobe is being degraded in lysosomes, since it is being taken up by endocytosis. This would not explain the observation that the FRET efficiency is higher in vivo than in vitro, however. It would predict the opposite.
The second concern is that you are measuring the FRET efficiency in vitro and in vivo by two different methods (spectrophotometer versus flow cytometer). These methods may not be comparable.
A third concern is that the results are inconsistent.
Finally, have you validated the bioprobe in vitro using purified caspase-3?
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