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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

Addgene's a Nonprofit? Nonprofit Awareness Day 2017

Posted by Joanne Kamens on Jun 5, 2017 11:19:13 AM

When you think of a "nonprofit" organization what do you think of? Maybe the term brings to mind a social service organization like the Red Cross or the American Cancer Society, or maybe you think about a local food pantry or community arts organization. Many people are surprised to learn that Addgene is officially filed, recognized and operated as a nonprofit under the United States Internal Revenue Code 501(c)(3). That means we were formed to benefit the public, not private interests. On this nonprofit awareness day, we layout the ways in which we promote our mission and work to enable researchers around the world.

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Topics: Inside Addgene

Beginner's Guide to Viral Vectors

Posted by Leila Haery on Jun 2, 2017 10:30:00 AM

You can use viral vectors for many experimental purposes. To help you make sense of all the viral vector information that's out there, Addgenie Leila Haery has summed up some of the most important characteristics of retroviruses, lentiviruses, AAVs, and adenoviruses in this easy-to-use guide. Print out the guide and use it for quick reference when you're designing your next virus experiment.

Subscribe to Viral Vector Blog Posts from Addgene

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Topics: Viral Vectors

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

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