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Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

Posted by Guest Blogger on Aug 27, 2020 9:15:00 AM

This post was contributed by Patrick Miller-Rhodes from the University of Rochester Medical Center. 

You’ve probably heard of Forster Resonance Energy Transfer (FRET). Through the non-radiative transfer for energy between neighboring fluorophores, FRET can be used to detect the inter- and intramolecular interactions that underlie protein function. However, FRET experiments can be difficult to implement in practice because FRET depends on a number of hard-to-achieve factors. For example, FRET requires that fusion proteins be in close proximity and present in large enough quantities (and the correct stoichiometric ratios) to generate useable data. What’s more, measuring and quantifying FRET is often easier said than done.

Fortunately, a complementary method exists for visualizing protein-protein interactions (PPIs): Bimolecular Fluorescence Complementation (BiFC).

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Topics: Fluorescent Proteins, FRET

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: Fluorescent Proteins, FRET, Fluorescent Biosensors

Technologies Enabled by NanoLuc® Luciferase

Posted by Guest Blogger on Feb 8, 2018 7:17:02 AM

This post was contributed by Kyle Hooper at Promega.

Researchers have been sharing plasmids ever since there were plasmids to share. Back when I was in the lab, if you read a paper and saw an interesting construct you wished to use, you could either make it yourself or you could “clone by phone”. One of my professors was excellent at phone cloning with labs around the world and had specific strategies and tactics for getting the plasmids he wanted. Addgene makes it so much easier to share your constructs from lab to lab. Promega supports the Addgene mission statement: Accelerate research and discovery by improving access to useful research materials and information. Many of our technology platforms like HaloTag® Fusion Protein, codon-optimized Firefly luciferase genes (e.g., luc2), and NanoLuc® Luciferase are available from the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

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Topics: Fluorescent Proteins, FRET

Special Delivery: Fluorophore Targeting for FRET Studies

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

This post was contributed by guest blogger James D. Fessenden, an Assistant Professor at Brigham and Women’s Hospital.

Biochemists often struggle to understand how a protein of interest actually behaves. How large is it? What parts of it move when you feed it substrate or add an essential cofactor? How many binding partners does it have and how do they come off and on in a cellular environment? If these are pressing issues in your laboratory, then FRET experiments are a viable biophysical path to answers.

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Topics: Fluorescent Proteins, FRET

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: Fluorescent Proteins, FRET, Fluorescent Biosensors

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