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. 2017 Mar 5;7(5):e2156.
doi: 10.21769/BioProtoc.2156.

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Nikos Kourtis  1 , Nektarios Tavernarakis  2
Affiliations

Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)

Nikos Kourtis et al. Bio Protoc. .

Abstract

Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

Keywords: Caenorhabditis elegans; FRAP; Messenger RNA; Protein synthesis; Protein translation.

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Figures

Figure 1.
Figure 1.. Photobleaching and recovery of fluorescence in vivo.
Representative images of roller, transgenic animals expressing pife-2GFP throughout somatic tissues, in a wild type or IFE-2 deficient background before photobleaching, immediately following an 8 min whole-animal photobleaching session, and after a 5 h recovery period, in the absence (a) and presence (b) of cycloheximide (500 μg/ml). Scale bars = 100 μm.
Figure 2.
Figure 2.. Regression analysis of fluorescence recovery in both wild type and IFE-2 deficient animals expressing pife-2GFP throughout somatic tissues.
Best-fit lines are generated for average pixel intensity values obtained during the recovery phase for the indicated genetic backgrounds (a, wild type; b, ife-2[ok306]; black lines). The respective equations describing best-fit lines as well as R2 values for each line are also shown. Line slope corresponds to the first derivative of fluorescent change within a time unit (Δf/dt), which is a measure of the recovery rate. Cycloheximide treatment (CHX; 500 μg/ml) results in negligible recovery rate (blue lines).
See this image and copyright information in PMC

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