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Tips for Using FRET in Your Experiments

Posted by Benoit Giquel on Nov 5, 2014 10:41:00 AM

The first time I heard about FRET during a journal club, my guitarist brain automatically thought about the raised element found on the neck of my guitar...not really useful for a biologist you would say. The student was of course talking about the now well-known FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules' interactions, modifications or dissociations in situ. Used since the mid-90s, this technique has revolutionised the way we apprehend molecular complexes and is still a very useful tool.   

Like a guitar hero (that I’m not), FRET loves playing “live”. Indeed, FRET was one of the first techniques which enabled the measurement of single molecule interactions in living cells using a microscope. Historically, molecular interactions were detected by indirect means often using probes with the potential to target several molecules. By analogy, it was like pointing out a group of students in a university hall but not knowing if these students know or interact with each other. FRET reduced the scale of our perception about molecular interactions.

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Topics: Imaging, Fluorescent Proteins

Choosing Your Fluorescent Proteins for Multi-Color Imaging

Posted by Guest Blogger on Oct 9, 2014 11:00:00 AM

This post was contributed by Kurt Thorn of the Nikon Imaging Center at UCSF.

A common requirement for live cell imaging experiments is the ability to follow multiple fluorescently tagged species simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation and emission spectra differ sufficiently for them to be imaged in distinct fluorescent channels on the microscope. With the proliferation of fluorescent proteins in recent years, there are many fluorescent protein combinations that can be imaged together, but this also means that the choice of fluorescent proteins requires some thought.

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Topics: Lab Tips, Fluorescent Proteins

The Michael Davidson Collection: One-Stop Shop for Fluorescent Proteins

Posted by Kendall Morgan on Jul 29, 2014 2:16:00 PM

In the world of fluorescent proteins and their use for imaging cell biology, Michael Davidson’s lab at Florida State University has been the go-to place. In 2012, his National High Magnetic Field Lab worked with an impressive 1,350 scientists from more than 275 institutions all over the world. In the course of all those collaborations over the years, he and his colleagues built a Molecular Expressions collection including some 3,300 plasmids along with image galleries and educational resources to go with them. This collection of plasmids is available in an easily searchable format on Addgene’s Michael Davidson Fluorescent Protein Collection webpage. (Check back regularly as new plasmids are being added to the webpage every week.)

Over 300 Backbones in a Rainbow of Colors

“It’s a great collection with over 300 backbones alone,” said Addgene’s Lianna Swanson, who has been working with members of the Davidson lab to coordinate the impressively big deposit. “He has every fluorescent protein under the sun, from the standard oldies but goodies (e.g., EGFP and YFP) to the new and improved fruit colors (e.g., apple, papaya, and tomato) and the photoactivatable fluors (e.g. Phamret and Dendra). It’s just such a great collection with such variety.”

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Topics: Hot Plasmids, Fluorescent Proteins

Which Fluorescent Protein Should I Use?

Posted by Guest Blogger on May 20, 2014 10:06:00 AM

This post was contributed by Gal Haimovich of greenfluorescentblog.

Be honest.  Do you really know how fluorescent proteins glow?  

Fluorescent Proteins (FPs) were first discovered over 50 years ago, with the discovery of the Green Fluorescent Protein (GFP), a protein from the jellyfish Aequorea Victoria. Since that discovery, the family of FPs just keeps getting larger with hundreds of variants available. Read on to familiarize yourself with the available FP emission colors and 10 points to keep in mind when choosing an FP (or two) for your upcoming experiments.

Fluorescence is the emission of light by a substance that has absorbed light. The emitted light is at a longer wavelength than the exciting wavelength. Thus, FPs are proteins with this unique capacity.

Many of these FPs are fluorescent when ectopically expressed in most organisms. Furthermore, fusing FPs to another protein usually does not affect its fluorescence. Therefore, FPs are used to study many biological questions. The two most common uses are: 1) to test the expression level in a specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to the protein of interest), thus tracking the localization of that biomolecule inside living cells.

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Topics: Plasmid How To, Plasmid Technology, Fluorescent Proteins

Plasmids 101: Green Fluorescent Protein (GFP)

Posted by Marcy Patrick on May 15, 2014 11:33:00 AM

Bioluminescence and fluorescence from proteins such as Green Fluorescent Protein (GFP) has likely existed in creatures such as jellyfish for millions of years; however, it took until the 1960s for scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives are a staple in the lab. GFP is used in research across a vast array of biological disciplines and scientists employ GFP for a wide number of functions, including: tagging genes for elucidating their expression or localization profiles, acting as a biosensor or cell marker, studying protein-protein interactions, visualizing promoter activity, and much more.

Read on to learn more about GFP, how scientists have evolved this versatile protein to suit their experimental needs, and some of the common applications in the lab.

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Topics: Plasmid Elements, Plasmids 101, Fluorescent Proteins

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