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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

Hot Plasmids: FRET-Based Biosensors

Posted by Kendall Morgan on May 6, 2014 9:07:55 AM

Oliver Griesbeck of the Max Planck Institute for Neurobiology has been working on genetically encoded indicators of calcium and other small molecules since the very beginnings of the field. Those engineered sensors were designed to replace synthetic calcium dyes, which had been in use since the 1980s.

“Synthetic dyes were the standard in the field, but there is one problem: how to get that into the cells of interest,” Griesbeck said. Because they are chemical compounds, they have to be applied or injected, and they don’t always end up where you want them to go.

Griesbeck is motivated by a particular interest in monitoring the activity and biochemistry of living neurons in an effort to understand the connection between molecular- and cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience.” 

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Topics: Plasmid Technology, Hot Plasmids, Fluorescent Proteins

In Living Color: The Skinny on In Vivo Imaging Tools

Posted by Kendall Morgan on Mar 27, 2014 1:14:57 PM

If you start poking around on Addgene’s Fluorescent Protein Guide to In Vivo Imaging, you’ll pretty quickly notice the name Vladislav Verkhusha popping up again and again, and for good reason.

We all know scientists have used fluorescent proteins to observe what’s happening inside cells for at least a couple of decades. Green is the classic color, but fluorescent proteins are available in a variety of hues. While those tools are great for many applications, Verkhusha and his lab at Albert Einstein College of Medicine in New York recognized their limitations for peering right through living animals to see their organs – a liver or brain, say, or maybe a tumor. They wanted to find something better.

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Topics: Plasmid Technology, Hot Plasmids, Imaging

Plasmids 101: Mammalian Vectors

Posted by Marcy Patrick on Mar 25, 2014 11:15:00 AM

Although plasmids do not naturally exist in mammals, scientists can still reap the benefits of plasmid-based research using synthetic vectors and cultured mammalian cells. Of course, these mammalian vectors must be compatible with the cell type they are tranfected into – a bacterial origin of replication (ORI) will not allow for plasmid replication in mammalian cells, for example, and a toxin that kills bacteria may not have any discernable effect on mammalian cells. In this blog post we will discuss how mammalian plasmids differ from their bacterial counterparts, including how replication occurs and whether selection is necessary for transfected cells.

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Topics: Plasmid How To, Plasmid Technology, Plasmids 101

CRISPR-Cas9 FAQs Answered!

Posted by Caroline LaManna on Mar 13, 2014 12:08:00 PM

As Kendall mentioned in Tuesday's blog post, keeping up with the newest CRISPR technologies and their applications can be exhausting. A quick search for "CRISPR", short-hand for Clustered Regularly Interspaced Short Palindromic Repeats, in Pubmed returned 728 articles (3/12/2014). With so many options for CRISPR plasmid tools and numerous experimental design decisions to make, it makes sense that scientists, many of whom are venturing into genome editing for the first time, have lots of questions.

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Topics: Plasmid Technology, Genome Engineering, Lab Tips, CRISPR

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