A BiT or BRET, Which is Better?

Now that Promega is expanding its offerings of options for examining live-cell protein interactions or quantitation at endogenous protein expression levels, we in Technical Services are getting the question about which option is better. The answer is, as with many assays… it depends! First let’s talk about what are the NanoBiT and NanoBRET technologies, and then we will provide some similarities and differences to help you choose the assay that best suits your individual needs. Continue reading “A BiT or BRET, Which is Better?”

NanoBRET™ Target Engagement Intracellular Kinase Assay Nominated for Scientists’ Choice Award®

Joins Nominees for Best New Drug Discovery & Development Product 2017

SelectScience® nominates NanoBRET™ Target Engagement Kinases Assay as a Best New Drug Discovery & Development product for 2017.

We were honored recently to have NanoBRET™ Target Engagement Intracellular Kinase Assays nominated by SelectScience® as one of the Best New Drug Discovery & Development Products of 2017. This is a Scientists’ Choice Award®, an opportunity for scientists like you worldwide to vote for your favorite new drug discovery/development product.

We are super excited about both the nomination and the NanoBRET™ Target Engagement Intracellular Kinase Assay. Here is a little information about the assay.

Continue reading “NanoBRET™ Target Engagement Intracellular Kinase Assay Nominated for Scientists’ Choice Award®”

Top 5 Most Read Promega Papers in 2017

It’s always nice to know that someone is reading your paper. It’s a sign that your research is interesting, useful and actually has an impact on the scientific community. We were thrilled to learn that papers published by Promega scientists made the top 10 most read papers of 2017 in the journal ACS Chemical Biology. In fact, Promega scientists authored five of the top six most read papers! Let’s take a look at what they are.

#5 CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide

Publication Date (Web): September 11, 2017

This 2017 paper introduces our newest star: HiBiT, a tiny 11aa protein tag. To any scientist studying endogenous protein expression, the HiBiT Tagging System is your dream come true. It combines quantitative and highly sensitive luminescence-based measurement with a tiny-sized tag that can be easily inserted into endogenous protein via CRISPR/Cas9 gene editing with little impact on native protein function. The HiBiT Tagging System has been listed as a 2017 Top 10 Innovation by The Scientist, and it will drastically change how we study endogenous protein expression. Continue reading “Top 5 Most Read Promega Papers in 2017”

Promega Partnering with UC-Davis Drought-Resistant Rice Project

The Foundation for Food and Agriculture Research (FFAR) announced on November 30 that they are awarding $1M to a project based at the University of California, Davis, to study protein kinases of rice plants. The team is led by Dr. Pamela Ronald, a leading expert in plant genetics who has engineered disease- and flood-resistant rice. This project aims to address the growing agricultural problem of water scarcity by gaining a better understanding of the role kinases play in enabling drought-resistance. Promega will be supporting this research by providing NanoBRET™ products to help characterize kinase inhibitors.

Principal Investigator Pamela Ronald, Ph.D. Photo Credit: Deanne Fitzmaurice

The research team will begin by screening over 1,000 human kinase inhibitors to determine which ones do interact with the plant kinome and, if applicable, which kinase(s) they inhibit. Once the compound library has been established, the team will assess the inhibitors’ phenotypic effects on rice to identify kinases that, when inhibited, positively impact root growth and development. The long-term goal is to use these findings to engineer drought-resistant rice.

Continue reading “Promega Partnering with UC-Davis Drought-Resistant Rice Project”

Research Teams Demonstrate Bivalent Binding of a Novel Bromodomain Protein Inhibitor

13305818-cr-da-nanoluc-application_ligundToday’s blog is written by guest blogger Kristin Huwiler from our Cellular Analysis and Proteomics Group.

Two research collaborations, one in Europe and a second in the US, have just published in Nature Chemical Biology (1,2) on the identification of BET inhibitors (bi-BETs) that bind via a bivalent mechanism to both bromodomains of BRD4. These bivalent chemical inhibitors exhibit high cellular potency and affinity relative to their monovalent predecessors. By developing high-affinity ligands that engage both bromodomains simultaneously within BRD4, the authors illustrate a concept that may be applicable in the development of selective, potent ligands for other multi-domain proteins. Here we review the work presented in the Waring et al. paper using the Promega NanoBRET™ Technologies to characterize the mechanism of action of their bivalent probe.

The bromodomain and extraterminal (BET) sub-family are some of the most studied bromodomain-containing proteins (3). The BET subfamily of proteins contain two separate bromodomains. BRD4 is one well studied member of the BET sub-family. Several small molecule inhibitors that target BRD4 have been developed as potential therapeutics for various cancers with promising initial studies (4), but to date are all monovalent, binding each bromodomain of the BET family members separately (2).
Continue reading “Research Teams Demonstrate Bivalent Binding of a Novel Bromodomain Protein Inhibitor”

Interrogating Protein Interactions: An Infographic for NanoBRET™ Assay Design

Yesterday my fellow blogger, Kari, posted a review of the ACS Chemical Biology paper describing a new BRET platform for analyzing protein-protein interactions. If you are interested in studying induction and inhibition of protein interactions in real time, take a look at the infographic below to learn how to develop a NanoBRET™ Assay to monitor your protein of interest.

21421668-IG-CR-NanoBRET-Infographic-Final

About the Development of an Improved BRET Assay: NanoBRET

"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

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”

Uncovering Protein Autoinhibition Using NanoBRET™ Technology

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”

Detecting Inhibition of Protein Interactions in vivo

Protein Interactions with NanoBRETIn a paper published in the September issue of ACS Medicinal Chemistry Letters, researchers from GlaxoSmithKline in the UK and Germany report on the discovery, binding mode and structure:activity relationship of a new, potent BRPF1 (bromodomain and PHD finger containing protein family) inhibitor. This paper came to our attention as it is one of the first publications to apply Promega NanoBRET technology in an vivo assay that reversibly measures the interaction of protein partners. The technology enabled the identification of a novel inhibitor compound that disrupts the chromatin binding of this relatively unstudied class of bromodomain proteins.

What exactly are bromodomains and why do they matter?
Bromodomains are regions (~100 amino acids) within chromatin regulator proteins that recognize and “read” acetylated lysine residues on histones. These acetylated lysines act as docking stations for regulatory protein complexes via binding of the bromodomain region. Because of their role in chromatin binding and gene regulation, bromodomains have attracted interest as potential targets for anti-cancer treatments. Although some bromodomain-containing proteins (e.g., those in the bromodomain and extraterminal domain (BET) subfamily) are well characterized and have been identified as potential therapeutic targets, others are less well understood. Continue reading “Detecting Inhibition of Protein Interactions in vivo”