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This post was contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology and van Leeuwenhoek Centre for Advanced Microscopy (University of Amsterdam).

Tagging a protein of interest with a fluorescent protein to study its function is one of the most popular applications of fluorescent proteins. These fusion proteins enable the observation of proteins in living cells and organisms. Both components of the chimera are encoded by DNA. Since researchers can generate almost any DNA sequence in the way that they like, the design and engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping all of the native properties of the protein of interest can be challenging. In this blog I discuss strategies to generate fusion proteins and highlight some aspects of their design. 

This post was contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology and van Leeuwenhoek Centre for Advanced Microscopy (University of Amsterdam).

GFP is the most popular, most widely used genetically encoded fluorescent probe. Several factors contribute to the popularity of GFP including (i) fast and complete maturation to functional, fluorescent protein in almost all organisms and cell types, (ii) no need to add a co-factor, (iii) easy visualization with standard filter sets on a fluorescence microscope, and finally (iv) good toleration in fusion proteins.

Since GFP is such a well-validated, all-round good performing probe, it is the first choice when selecting a genetically encoded fluorescent tag. There are, however, a number of limitations that you may run into if you choose to use it. Several of these limitations and possible solutions are discussed below.

In complex metazoans, rapid cell division and large scale cell mobility are essential processes during embryonic development. These are required for a growing organism to make the complicated transition from a clump of cells to a fully differentiated body. In contrast, these dynamic processes are largely absent in adult organisms, where tissues structures are more stable and local movements predominate (e.g. a basal progenitor cell migrating to the epithelium). At this stage, only cells from the immune system show wide scale mobility with movement from the bone marrow and other lymphoid organs to specific tissues where they can scan for any signs of danger. In this post we’ll focus on how fluorescent proteins can and have been used to monitor cellular movements in the immune system. The techniques used here could be adapted to studying other systems in which there is large scale cellular movement throughout an organism.

Luminescent molecules are very useful tools because we can easily detect and measure the light they emit. Proteins that give off light include chemiluminescent proteins, like luciferases, as well as fluorescent ones, like Green Fluorescent Protein (GFP). These molecules occur naturally in bioluminescent organisms, but their real power lies in the clever ways sceintists have adapted them for use in the laboratory.

Imagine being able to determine whether two proteins are within 10 nanometers of each other, or measure the tension in the helical structure of spider silk, or the activity of a protein in a synapse. What kinds of tools enable us to measure these properties, and what fascinating experiments could push these tools even further? All of these things can be done using FRET! Read on to find out more about this amazing imaging technique and find further tips for using FRET in your experiments here.