Can anyone please suggest to me whether I can able to check the fret efficiency between two proteins when one protein expressed after transfecting the cells, that might act as a donor( FITC-tagged), and the other protein expressed using antibody staining, which acts as an acceptor (mcherry-tagged)?
Staining/expressing the two proteins by two different techniques will make any difference in measuring the FRET-FLIM efficiency or not?
A few things to consider:
- specificity of the Ab used
- introduction of Ab into cells and toxicity
- Ab-based sensors are not stable in living cells and can be quickly cleared away from the body in vivo
- Half-life of transfected protein and that of the anti-body
I recommend you go through the following articles to help you decide:
- A Guide to Fluorescent Protein FRET Pairs. Sensors 2016, 16, 1488 (a good review which discusses the above points)
- Protein localization in living cells and tissues using FRET and FLIM. Differentiation (2003) 71:528–541.
If you think that you want to go the transfection-Ab labeling mode for your studies:
- Labeling proteins inside living cells using external fluorophores for microscopy. Teng et al. eLife 2016;5:1-13 (for Ab labeling of live cells)
Good luck.
@Sathya Srinivasan
I believe FTIC is 488/505 excitation emission and the mCherry is 640/655 excitation emission. This will not be the quite good pair for FRET experiment. Do you have any dye which can be excite with 561 laser instead. For example Cy3 is 555/566 excitation and emission. This can be aired with either FTIC or with mCherry as a FRET pair more successfully.
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