ISSN: 2155-9872

Journal of Analytical & Bioanalytical Techniques
Open Access

Our Group organises 3000+ Global Conferenceseries Events every year across USA, Europe & Asia with support from 1000 more scientific Societies and Publishes 700+ Open Access Journals which contains over 50000 eminent personalities, reputed scientists as editorial board members.

Open Access Journals gaining more Readers and Citations
700 Journals and 15,000,000 Readers Each Journal is getting 25,000+ Readers

This Readership is 10 times more when compared to other Subscription Journals (Source: Google Analytics)
  • Research Article   
  • J Anal Bioanal Tech 2017, Vol 8(6): 390
  • DOI: 10.4172/2155-9872.1000390

tFRAP: A FRAP-Based Technique to Monitor Protein Translation in Living Cells

Nadine Griesche1,2, Verena Pesch1, Rohit Nalavade1, Stephanie Weber1, Ireen König1, Manuel Schölling1, Christoph Möhl1 and Sybille Krauß1*
1German Center for Neurodegenerative Diseases (DZNE), , Sigmund-Freud-Str.27, 53127 Bonn, Germany
2Department of Medical Cell Biology, Uppsala University, BMC, Box 571, 75123 Uppsala, Sweden
*Corresponding Author : Sybille Krauß, German Center for Neurodegenerative Diseases (DZNE), Sigmund-Freud-Str.27, 53127 Bonn, Germany, Tel: +4922843302555, Email: sybille.krauss@dzne.de

Received Date: Nov 27, 2017 / Accepted Date: Dec 04, 2017 / Published Date: Dec 18, 2017

Abstract

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.

Keywords: Translation; mRNA; Live cell; Flourescence recovery after photobleaching

Citation: Griesche N, Pesch V, Nalavade R, Weber S, König I, et al. (2017) tFRAP: A FRAPBased Technique to Monitor Protein Translation in Living Cells. J Anal Bioanal Tech 8: 390. Doi: 10.4172/2155-9872.1000390

Copyright: © 2017 Griesche N, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Top