| Popis: |
Detecting when and how much a protein molecule is synthesized is important for understanding cell function, but current methods have poor cellular or temporal resolution or are destructive to cells (Dahm et al., 2008). Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use split red fluorescent protein to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel and arbitrarily reset the time of detection of synthesis events, continually over time. |
