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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, Choosing the Brightest Fluorescent Protein

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, Choosing the Brightest Fluorescent Protein

A Practical Approach to Choosing the B(right)est Fluorescent Protein

Posted by Guest Blogger on Jun 1, 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.

Before you decide which car you want to buy, it is worthwhile to test-drive a couple of candidates. Before you buy a new microscope, it is smart to look at (and through) a couple of models. Before you start a new project with fluorescent proteins, the best advice is to try a couple of promising variants to check how they perform under your experimental conditions. This is time well spent and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts explains how to critically assess the reported properties of fluorescent proteins, how to do a head-to-head comparison of fluorescent proteins and how to make a well-informed decision on the best fluorescent protein for your application.

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Topics: Fluorescent Proteins, Choosing the Brightest Fluorescent Protein

Luminescent Imaging with Nano-lanterns

Posted by Mary Gearing on May 25, 2017 10:30:00 AM

Fluorescent imaging techniques have become indispensable tools for molecular and cell biologists over the last two decades, but their use can be limited by a few caveats. Since fluorescent proteins (FP) require external light activation, you can’t use fluorescence to monitor processes directly affected by light. Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be affected by both autofluorescence and photobleaching. Researchers have long been interested in using luminescence to get around these issues, but this solution wasn’t practical due to the low intensity of luminescent proteins. To make luminescent imaging a reality, Addgene depositor Takeharu Nagai and colleagues at Osaka University have developed Nano-lantern technology. Nano-lanterns contain a Renilla luciferase variant fused to an FP; when supplied with a luciferase substrate, the luciferase transfers energy to the FP, resulting in a fluorescent signal. Since their first publication in 2012, the Nagai laboratory has assembled a collection of multicolored nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion proteins.

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Topics: Hot Plasmids, Fluorescent Proteins

Plasmids 101: Fluorescent Biosensors

Posted by Jessica Welch on May 18, 2017 10:30:00 AM

Addgenie Mary Gearing contributed to the content of this article.

Biosensors (‘biological sensors’) are biological tools that monitor a process or detect a given molecule. The sensor component is usually a protein that undergoes a conformational change in response to the molecule it detects. This change then generates a reporter signal. Reporter signals may be electrochemical or light-based, with luminescent and fluorescent reporters being especially popular. We’ll give you an introduction to fluorescent biosensors, but keep in mind that there is a lot of variety in how biosensors work, and you should always check the associated publication for the specifics of your chosen plasmid.

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Topics: Fluorescent Proteins

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