When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

Posted by Guest Blogger on Apr 19, 2016 10:30:00 AM

This post was contributed by guest blogger Erik L. Snapp.

Stop using EGFP/GFP for fusion proteins! Despite multiple studies in high profile journal articles, many researchers remain unaware that EGFP/GFP is prone to forming noncovalent dimers. This property of EGFP can lead to significant artifacts.

If you're using green fluorescent protein or Enhanced Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label of cells, there's no problem. You're OK. However, if you fuse your protein of interest (POI) to GFP to study the protein's behavior in cells, in solution or something in between, you are using a tag with a serious drawback. The standard EGFP plasmid that used to be sold by Clontech and is in a freezer box in just about every lab in the world, is not inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means that your POI fused to GFP or another fluorescent protein (FP) could be forming dimers in cells. Why should you care? Three simple ways a dimeric FP could ruin your day (and experiment) are listed below. Solutions to avoid these all too common issues follow.

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Topics: Imaging, Fluorescent Proteins

Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

Posted by Mary Gearing on Nov 24, 2015 10:30:00 AM

At Addgene, we love GFP, and we’re always excited when depositors find new ways to make this workhorse protein even more useful! From FPs optimized for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things. Now, work from Connie Cepko’s lab allow researchers to activate transcription or Cre recombinase activity only in the presence of GFP. These systems, known as T-DDOG and Cre-DOG, respectively, repurpose popular GFP reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop new lines.

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Topics: Plasmid Technology, Synthetic Biology, Fluorescent Proteins, Cre-lox

Illuminating Epigenetics with A FRET Based Biosensor

Posted by Emma Markham on Nov 19, 2015 10:30:00 AM

Epigenetics has recently been hitting the headlines, with sotires like the potential devastation of the palm oil industry through epigenetic effects on the Cover of Nature. So what is epigenetics and what tools are available to study it?

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Topics: Hot Plasmids, Fluorescent Proteins, Techniques

Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

Posted by Guest Blogger on Oct 27, 2015 11:00:00 AM

 This post was contributed by guest bloggers Erik L. Snapp and Lindsey M. Costantini.

"You underestimate the power of the Dark Side."

--Darth Vader in "Return of the Jedi"

While Vader was referring to the evil side of a mystical "Force," this quote is equally applicable to many microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent, and misfolded FP-fusions on quantitative imaging experiments and cell physiology in general.

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Topics: Imaging, Fluorescent Proteins

Mapping the 4D nucleome with CRISPR/Cas9

Posted by Mary Gearing on Aug 11, 2015 10:30:00 AM

It seems that there’s a new CRISPR advance or technique published every week! One of the newest applications is a colorful system that uses fluorescently labeled Cas9 to label multiple genomic loci in live cells. While other systems can be used to label loci, such as fluorescence in situ hybridization (FISH) or fluorescently labeled TALEs, CRISPR/Cas9’s ease of use and ability to label live cells make this system truly advantageous. This new technique, developed in Thoru Pederson’s lab, brings us one step closer to mapping the 4D nucleome, the organization of the nucleus in space and time, and to understanding how nuclear organization varies across the life of a cell, or how organization may be altered in disease states.

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Topics: CRISPR, Imaging, Fluorescent Proteins

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