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Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

Posted by Guest Blogger on Apr 9, 2019 9:13:55 AM

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. 

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Topics: Fluorescent Proteins, Fluorescent Proteins 101

Fluorescent Proteins 101: When GFP lets you down

Posted by Guest Blogger on Aug 23, 2018 8:05:04 AM

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.

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Topics: Fluorescent Proteins, Fluorescent Proteins 101

History of Fluorescent Proteins

Posted by A Max Juchheim on Aug 7, 2017 9:58:40 AM

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.

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Topics: Fluorescent Proteins, Fluorescent Proteins 101

Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

Posted by Guest Blogger on Jun 15, 2017 10:30:00 AM

This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced microscopy, University of Amsterdam.

The previous two posts in this series described a practical approach to selecting a bright fluorescent protein and a photostable fluorescent protein. In the third post of this series, we will discuss how to select a non-aggregating fluorescent protein.

In the jellyfish Aequorea victoria, AvGFP forms a homodimer. In corals, the red fluorescent proteins form tetramers. In general, fluorescent proteins have a natural affinity and a tendency to form higher order aggregates. This property can be tolerated in some applications (e.g. labeling of cells or tracking promotor activity), but it is problematic in applications in which the fluorescent protein is used as an inert protein module. This is explained in more detail here. There are a variety of methods that can be used to measure your fluorescent protein’s propensity to aggregate. The basics and pitfalls of these experiments are discussed here.

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Topics: Fluorescent Proteins, Fluorescent Proteins 101

Choosing the B(right)est Fluorescent Protein: Photostability

Posted by Guest Blogger on Jun 8, 2017 10:30:00 AM

This post was contributed by guest bloggers Joachim Goedhart and Marieke Mastop from the Section of Molecular Cytology and Van Leeuwenhoek Centre for Advanced microscopy, University of Amsterdam.

The previous post in this series described a practical approach to selecting a bright fluorescent protein. In the second post of this series, we will discuss how to select a photostable fluorescent protein.

Photobleaching is the irreversible destruction of a fluorophore under the influence of light. Any fluorescent molecule will photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable. On top of photobleaching, fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017) and photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent proteis should emit a stable fluorescence signal, showing no or little deterioration or change of the signal during the course of the experiment.

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Topics: Fluorescent Proteins, Fluorescent Proteins 101

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