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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

Measuring Kinase Activity at the Single-Cell Level with Kinase Translocation Reporters (KTRs)

Posted by Beth Kenkel on Jul 26, 2018 8:46:55 AM

Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at least one site. Phosphorylation is particularly important for regulating signal transduction and measuring kinase activity at the single-cell level can aid in drawing connections between signaling activity and cell phenotype. One method for monitoring live single-cell kinase activity is FRET, but FRET reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with their Kinase Translocation Reporters (KTRs) whose cellular localization serves as a proxy measurement of kinase activity. The key advantage of KTRs is that they are easy to create and simple to multiplex.

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Topics: Hot Plasmids, Fluorescent Proteins

FPbase: A new community-editable fluorescent protein database

Posted by Guest Blogger on May 16, 2018 9:00:00 AM

This post was contributed by guest blogger Talley Lambert, a Research Associate at Harvard Medical School.

The need for a community fluorescent protein database

As recognized by the 2008 Nobel Prize, fluorescent proteins (FPs) have become one of the most indispensable tools in modern biological research.  Any microscopist will tell you that selection of a fluorescent probe (be it an organic dye or FP) is one of the most important steps in the design of an imaging experiment.  The choice is non-trivial, however, as FPs are tremendously complicated entities with a large range of characteristics (color, brightness, photostability, maturation, oligomerization), many of which are dramatically affected by environmental conditions (such as temperature, pH, fusion protein, etc...).  There are many online guides – including an excellent series of posts by Joachim Goedhart on the Addgene blog – outlining various important considerations when choosing a FP, but much of the primary data one might require when making such a decision remains spread across literature in publications that introduce these tools.

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Topics: Fluorescent Proteins, Scientific Sharing

Switch to GECO? An overview of AAV Encoded Calcium Sensors

Posted by Leila Haery on Apr 26, 2018 9:24:32 AM

As part of our partnership with the Penn Vector Core, we will be expanding our inventory of tools for calcium sensing. In this post, we’ll review the main categories of sensors we’ll have available.

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Topics: Viral Vectors, Fluorescent Proteins

Imaging Tools and Following a Passion for Basic Biology: Interview with Joachim Goεdhart

Posted by Tyler Ford on Mar 9, 2018 9:00:00 AM

Today on the Addgene Podcast, we have another interview conducted by European Outreach Scientist Benoit Giquel. Ben recently spoke with Joachim Goεdhart, a professor at the University of Amsterdam. In addition to creating and sharing many great fluorescent protein tools, Professor Goedhart is very active on Twitter and has helped us update many of our educational resources. Listen to hear all about Professor Goedhart, his lab, and some of the tools he’s developed.

 

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Topics: Podcast, Fluorescent Proteins

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