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

Posted by Tyler Ford 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

Important Considerations When Using AAVs

Posted by Guest Blogger on Jun 13, 2017 10:30:00 AM

This post was contributed by guest blogger Katrina Armstrong, a Neurophysiology Msc Student at the University of Manitoba.

  1. Location, Location, Location!
  2. Failure to Plan (for Storage) Is Planning to Fail
  3. Patience Is Bitter but Its Fruit Is Sweet
  4. The Future?

Need Virus? Check out Addgene's New Viral Service!

I knew little about adeno-associated Viral Vectors (AAVs) before starting my graduate program at the University of Manitoba. Our lab has been utilizing chemogenetics (Designer Receptors Exclusively Activated By Designer Drugs, DREADDs) and optogenetics as tools to investigate the roles of certain cell types in locomotion. We have relied heavily upon AAV vectors to deliver chemogenetic/optogenetic constructs into our cells of interest. Although they have a small packaging capacity, AAV vectors were suitable for our needs for the following reasons:

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Topics: Viral Vectors

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.

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Topics: Viral Vectors

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