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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

Anti-CRISPRs: Switching Off CRISPR-Cas9

Posted by Beth Kenkel on May 23, 2017 10:30:00 AM

CRISPR-Cas9 technology is constantly evolving. Variants of Cas9 can be used for genome editingactivating gene expression, repressing gene expression, and much more. But there’s one thing that’s been missing: a way to shut off Cas9’s activity after it’s been turned on. The concern is that the longer Cas9 remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas9 activity using light or drugs have been developed, the field lacked an “off-switch” for Cas9.

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Topics: CRISPR

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

New Podcast Segment: Hot Plasmids

Posted by Tyler Ford on May 17, 2017 11:37:42 AM

We’re breaking into more audio and video on the Addgene Blog and Addgene website. As we push forward with these efforts, you’ll find new ways to learn about science careers, lab protocols, and, of course, plasmids. Today we’re trying a new way to present plasmid info with a new segment on the Addgene podcast - The Hot Plasmids Segment. Click on the player below to listen to a quick (~5 min) Hot Plasmids podcast that introduces you to 4 new plasmid technologies from one of our recent newsletters.

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Topics: Hot Plasmids, Podcast

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