| Popis: |
Traditionally, studies on protein translation rely on systems, in which cells have been lysed prior determination of levels of the protein of interest. However, these assays do not reflect the protein synthesis in living cells in real time, but analyze protein levels after a given incubation time, leading to limitations in results based on experimental parameters. To overcome this problem, we have previously established a Fluorescence recovery after photobleaching (FRAP)-based technique to monitor protein translation in living cells. For this, the protein of interest fused to green fluorescent protein (GFP) is expressed in cell lines. After bleaching the entire cell, the fluorescent signal of the protein of interest is lost, allowing to capture the signal recovery of newly translated GFPtagged protein over time. Here we present two improved versions of this technique using different fluorescent dyes: tFRAP (translational FRAP). For the first improved version of tFRAP we have inserted a second fluorescent dye, red fluorescent protein (RFP), into the same expression vector that drives expression of the protein of interest fused to GFP driven by a second promoter. For the second improved version of tFRAP we have fused our protein of interest to a photo-switchable dye, Dendra2. Both improved versions allow to correct the fluorescence signal intensity of the protein of interest for different transfection rates of individual cells. These two advanced techniques are new powerful tools for quantifying translation rates in living cells and will be useful in future studies on mRNA translation. |
