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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

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: Fluorescent Proteins, FRET

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: Fluorescent Proteins, FRET, Fluorescent Biosensors

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