BRET

New Bioluminescent Sensor Can Detect Multiple Antibodies in a Single Drop of Blood

Posted on by Johanna Lee
nanoluc invivo imaging

Antibody tests are often used to determine whether individuals have been exposed to certain bacteria or viruses. For most existing antibody tests, the process goes something like this: A vial of blood is drawn from the individual, the vial is sent to a lab, then a trained technicians performs the antibody test and sends back the results. The current process is less than ideal for a few reasons. For one, blood draws are invasive and can be painful. Also, getting results could take days, due to the time required to deliver and process the sample. Lastly, costs can be high, since the need for trained professionals and specialized instruments in laboratory settings adds to the cost of each test.

What if all you needed to do for an antibody test was apply a single drop of blood onto a thin piece of film, and you would get results on the spot within five minutes? Scientists have recently developed an antibody test based on bioluminescent technology that could make this a reality. They describe their findings in a recent study published in ACS Sensors.

Continue reading “New Bioluminescent Sensor Can Detect Multiple Antibodies in a Single Drop of Blood”

NanoLuc® Luciferase Powers More than Reporter Assays

Posted on by Sara Klink
Bright NanoLuc® Luciferase

What can you do with a small, super bright luciferase? Amazing things. We’ve highlighted many of the papers and new applications that NanoLuc® luciferase has enabled on this blog. While NanoLuc® luciferase was first introduced as a reporter enzyme to assess promoter activity, its capabilities have expanded far beyond a genetic reporter, creating bioluminescent tools used to study endogeneous protein dynamics, target engagement, protein degradation, immunodetection and more. So where did the NanoLuc luciferase come from and how does one enzyme power so many research capabilities? Read further for a primer on the various technologies and applications developed from this enzyme over the last 10 years.

Continue reading “NanoLuc® Luciferase Powers More than Reporter Assays”

Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository

Posted on by Kyle Hooper

Researchers having 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 this 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 present in the repository. We encourage people to go to Addgene to get new innovative tools. Afterall, isn’t science better when we share?

I’d like to focus on some tools in the Addgene collection based on NanoLuc® Luciferase (NLuc).  The creation of NanoLuc® Luciferase and the optimal substrate furimazine is a good story (1).  From a deep sea shrimp to a compact powerhouse of bioluminescence, NLuc is 100-fold brighter than our more common luciferases like firefly (FLuc) and Renilla (RLuc) luciferase.  This is important not so much for how bright you can make a reaction but for how sensitive you can make a reaction.  NLuc requires 100-fold less protein to produce the same amount of light from a Fluc or RLuc reaction.  NLuc lets you work at physiological concentrations.  NLuc is bright enough to detect endogenous tagged genes generated through the CRISPR/Cas9 knock-in.  NLuc is very inviting for endogenous tagging as it is only 19kDa.  An example is the CRISPaint-NLuc construct (Plasmid #67178) for use in the system outlined in Schmid-Burgk, J.L. et al (2).

Two applications of NanoLuc® Technology have caught my attention through coupling the luciferase with fluorescent proteins to make better imaging reporters and biosensors. Continue reading “Shining Stars: Cool NanoLuc® Plasmid Constructs Available Through the Addgene Repository”

Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events

Posted on by Kyle Hooper

A Bioluminescent Alternative

Fluorescence resonance energy transfer (FRET) probes or sensors are commonly used to measure cellular events. The probes typically have a matched pair of fluorescent proteins joined by a ligand-binding or responsive protein domain. Changes in the responsive domain are reflected in conformational changes that either bring the two fluorescent proteins together or drive them apart. The sensors are measured by hitting the most blue-shifted fluorescent protein with its excitation wavelength (donor). The resulting emission is transferred to the most red-shifted fluorescent protein in the pair, and the result is ultimately emission from the red-shifted protein (acceptor).

As pointed out by Aper, S.J.A. et al. below, FRET sensors face challenges of photobleaching, autofluorescence, and, in the case of exciting cyan-excitable donors, phototoxicity. Another challenge to using FRET sensors comes when employing optogenetic regulators to initiate the event you wish to monitor. Optogenetic regulators respond to specific wavelengths and initiate signaling. The challenge comes when the FRET donor excitation overlaps with the optogenetic initiation wavelengths. Researchers have sought to alleviate many of these challenges by exchanging the fluorescent donor for a bioluminescent donor, making bioluminescence resonance energy transfer (BRET) probes. In the three papers described below, the authors chose NanoLuc® Luciferase as the BRET donor due to its extremely bright signal.

Continue reading “Making the Switch from FRET to BRET: Applications of NanoLuc® Luciferase with Fluorescent Protein Acceptors for Sensing Cellular Events”

About the Development of an Improved BRET Assay: NanoBRET

Posted on by Kari Kenefick

"Protein BRD4 PDB 2oss" by Emw - Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons - https://commons.wikimedia.org/wiki/File:Protein_BRD4_PDB_2oss.png#/media/File:Protein_BRD4_PDB_2oss.png
“Protein BRD4 PDB 2oss” by Emw – Own work. Licensed under CC BY-SA 3.0 via Wikimedia Commons – https://commons.wikimedia.org/wiki/File:Protein_BRD4_PDB_2oss.png#/media/File:Protein_BRD4_PDB_2oss.png

One of the more exciting reporter molecules technologies available came online in the past year, with the launch of the Promega NanoBRET™ technology. While it’s easy for me, a science writer at Promega, to brag, seriously, this is a very cool protein interactions tool.

A few of the challenges facing protein-protein interactions researchers include:

  • The ability to quantitatively characterize protein-protein interactions
  • Ability to examine protein-protein interactions in situ, in the context of the living cell

A goal of the NanoBRET™ developers was to improve the sensitivity and dynamic range of traditional BRET technology, in order to address these challenges.

In May 2015 these researchers published an article outlining their efforts to create NanoBRET technology in ACS Chemical Biology, in an article entitled, “NanoBRET—A Novel BRET Platform for the Analysis of Protein-Protein Interactions”. Here is a brief look at their work.

Continue reading “About the Development of an Improved BRET Assay: NanoBRET”

If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions

Posted on by Michele Arduengo

14231183 WB MS Protein Interactions Hero Image 600x214Robert Hooke first coined the term “cell” after observing  plant cell walls through a light microscope—little empty chambers, fixed in time and space. However,  cells are anything but fixed.

Cells are dynamic: continually responding to a shifting context of time, environment, and signals from within and without. Interactions between the macromolecules within cells, including proteins, are ever changing—with complexes forming, breaking up, and reforming in new ways. These interactions provide a temporal and special framework for the work of the cell, controlling gene expression, protein production, growth, cell division and cell death.

Visualizing and measuring these fluid interactions at the level of the cell without perturbing them is the goal of every cell biologist.

A recent article by Thomas Machleidt et al. published in ACS Chemical Biology, describes a new technology that brings us closer to being able to realize that goal. Continue reading “If We Could But Peek Inside the Cell …Quantifying, Characterizing and Visualizing Protein:Protein  Interactions”

Targeting MYC: The Need to Study Protein:Protein Interactions in Cells

Posted on by Michele Arduengo

Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;
Crystal Structure of MYC MAX Heterodimer bound to DNA ImageSource=RCSB PDB; StructureID=1nkp; DOI=http://dx.doi.org/10.2210/pdb1nkp/pdb;

In 1982, picked up because of its homology to chicken virus genes that could transform cells, MYC became one of the first human genes identified that could drive cellular transformation (1,2). Since that time countless laboratories have prodded and poked the human MYC gene, the MYC protein, their homologs in other animal models, and their transforming viral counterparts.

MYC is a transcription factor and forms heterodimers with a required protein partner, MAX, before binding to the E box sequences of DNA regulatory regions (3). MYC regulates gene expression of many targets through interactions with a host of proteins, often referred to as the MYC Interactome (2).  In fact, MYC is estimated to bind 10–15% of the genome, and it regulates the expression of genes that  are transcribed by by each of the three RNA polymerases (2).

MYC plays a central role in regulating cell growth, proliferation, apoptosis, differentiation and transformation, acting as a central integrator of cellular signals. MYC is tightly regulated at multiple levels from gene expression to protein stability. Dysregulation (usually upregulation) of the amount and stability of Myc protein is observed in many human cancers. Even in cancers in which MYC is not directly involved in transforming cells, its normal expression is often required to support the extracellular matrix and/or vascularization necessary for tumor growth and formation (4).

Because MYC is such a central player cancer pathology, it is an attractive target for cancer therapeutics  (2) . Continue reading “Targeting MYC: The Need to Study Protein:Protein Interactions in Cells”

Uncovering Protein Autoinhibition Using NanoBRET™ Technology

Posted on by Sara Klink

13305818-protein ligandIntroducing new assays or technologies is meant to make it easier for you to perform research and craft experiments to test hypotheses. However, scientists are creative people, and new technologies or assays may just be the catalyst for a crucial experiment or new data you are seeking. In the case of a recent Proceedings of the National Academy of Sciences USA article, Wang et al. used the principle of our NanoBRET™ assay to understand how ERK1/2 phosphorylation of Rabin8, a guanine nucleotide exchange factor, influenced its configuration and subsequent activation of Rab8, a protein that regulates exocytosis. Continue reading “Uncovering Protein Autoinhibition Using NanoBRET™ Technology”

Tools for Observing Protein Behaviors Inside Cells

Posted on by Michele Arduengo

nanoluchomepageimageIn this BioCompare video, Promega scientist, Dr. Keith Wood, explains how the smaller, brighter and sensitive NanoLuc® Luciferase allows scientists to observe protein behaviors inside cells, including rare events. See how a luciferase can now be used to investigate protein activities as well as for traditional luciferase genetic reporter assays.

http://www.biocompare.com/Life-Science-Videos/156795-Tools-for-Analysis-of-Protein-Behavior-In-Living-Cells/?arev=true