Discover Glo Tour 2017

Join one of our free scientific symposia at

Discover Glo
European Tour 2017

27 - 31 March 2017 | 3 - 7 April 2017

Explore innovation on a molecular scale in 8 different cities across Europe.

Attend a symposium at a city near you and discover how new luminescent technologies are improving reporter assays and allowing researchers to study protein interactions and target engagement in real time in live cells.

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Thomas Machleidt, Ph.D.

Senior Scientist at Promega Corporation

Dr. Machleidt currently leads the cell biology team within the Advanced Technology Group. His main interest is the development and application of novel technology platforms for the analysis of protein dynamics in cells. Dr. Machleidt earned his M.S. in Biology from the Eberhardt Karls University in Tuebingen. He received his Ph.D. in Immunology for his work on the signaling mechanisms of the pro-inflammatory cytokine TNF in Prof. Kronke’s lab at the Technical University of Munich. For his postdoc he joined the lab of RGW Anderson at UT Southwestern Medical Center where he investigated the structure and function of the caveolin protein family. He subsequently held R&D positions at Johnson & Johnson and Ansata Therapeutics. Prior to joining Promega in 2011 he worked as a group leader for 7 years at Life Technologies. Dr. Machleidt has authored and co-authored 48 peer reviewed publications and book chapters.

Topics & Abstracts

One of the key challenges of the post-genomic area is the characterization of protein expression and function in the appropriate physiological context. One key mechanism for controlling protein function is dynamic regulation of protein abundance either through changes in expression or protein stability. Current technologies for the quantitative analysis of endogenous protein levels are heavily dependent on immune-detection, which is time consuming and often limited by availability and quality of antibodies.

Here we introduce a novel method, HiBiT, a multifunctional tag that allows rapid tagging of endogenous proteins via CRISPR/Cas9 mediated gene-editing. The HiBiT tag was developed as part of the NanoLuc Binary Technology (NanoBiT®), a complementation system based on NanoLuc® luciferase. The 11 amino acid HiBiT peptide and its complementing 18 kDa polypeptide, known as large BiT (LgBiT), spontaneously reconstitute into an active luciferase derived from the NanoLuc enzyme. The HiBiT assay system provides over 7 logs of linear dynamic range with a LOD of less than 0.1 attomoles, which makes this technology unique for the quantitative analysis of endogenous proteins.

The broad utility of combining HiBiT technology with CRISPR-mediated gene editing is exemplified by the rapid generation of multiple cell-based assays that allowed the analysis of protein abundance of multiple proteins along the hypoxia pathway in response to treatment with pathway modulating compounds. Furthermore this presentation will introduce additional HiBiT applications for protein analysis including bioluminescence imaging, target engagement and measurement of posttranslational modifications using a variety of biological models.

All aspects of protein function and activity are governed by an intricate network of interactions between proteins and other molecular constituents. Capturing the dynamic nature of those interactions and their physiological relevance requires the development of technologies that allow interaction analysis in living cells.

Here we present two novel technology platforms, NanoBRET and NanoLuc Binary Technology (NanoBiT) that enable the investigation of molecular interactions of proteins with unprecedented sensitivity in living cells. Both methods rely on NanoLuc®, a small (19 kDa) luciferase enzyme engineered for structural stability and the ability to generate an intense, steady, bioluminescent signal. NanoBRET was conceived as novel bioluminescence resonance energy transfer (BRET) platform using NanoLuc as donor paired with a red fluorescent acceptor.

The NanoBiT complementation is comprised of a pair of complementary subunits (18 and 1 kDa) which exhibit very low self-association and yet can efficiently reconstitute bright luminescence activity when brought into close proximity. This presentation will provide a comprehensive overview of both technologies focusing on a wide variety of biological models and applications that illustrate the versatility of NanoBRET and NanoBiT for quantitative analysis of protein-protein and protein-small molecule interactions in living cells.

Real-time continuous monitoring of cellular processes offers distinct and important advantages over traditional end-point assays. A comprehensive representation of the changes occurring in live cells over time informs about the biological status of the cell, and enables decision making about the timing of treatments or the use of other functional end-point assays. The ability to measure real time change is particularly valuable for the analysis of cell death where changes in viability are highly dependent on treatment, molecular mechanism and cellular background.

This presentation will provide an introduction to a new generation of live-cell assays that allow the continuous measurement of different parameters of cell health and apoptosis. RealTime-Glo™ MT cell viability assay uses the reducing potential of the cell as a measure for cell health. A wide range of applications for the RealTime-Glo MT assay will be discussed.

The RealTime-Glo™ Annexin V Apoptosis Assay represents the first homogenous reagent that allows real time measurement of annexin V binding to phosphatidylserine (PS), a well-established hallmark of the apoptotic phenotype, in live cells. In addition we will combine the RealTime-Glo Annexin V assay with bioluminescence imaging to follow the induction of apoptosis and Natural Killer Cell--mediated cytotoxicity at the single-cell level.

Drug action is generally dependent on engagement of a protein target within its physiological context. The selectivity, affinity and kinetic of this binding event is critical for efficacy and is in turn influenced by the complex physiological environment encountered in vivo. We present the first technique to measure target engagement under physiological conditions in living cells. NanoBRET enables quantitative biophysical assessment of compound engagement and residence time for a chosen intracellular target.

Using protein kinases as example we will show how this HTS-compatible method enables analysis of isozyme-specific affinity and binding kinetics over entire enzyme classes. Additional case studies will demonstrate how this technique can be utilized to study mechanistic aspects of intracellular target engagement including the influence of cellular ATP concentration on kinase affinity profiles and the importance of intracellular residence time for kinetic selectivity of kinase inhibitors.

8 Locations in 8 Days.
Choose your location to register and learn more.

Mon. 27 March 2017

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Tue. 28 March 2017

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Wed. 29–31 March 2017

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Mon. 3 April 2017

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Promega United Kingdom

Hilary Latham
E: hilary.latham@promega.com
P: 02380 717 319

Promega Benelux

Lindsay Mesure, Product Specialist Promega BNL
E: lindsay.mesure@promega.com
P: +32(0)473 37 55 59

Promega Switzerland

Réka Nagy
E: reka.nagy@promega.com
P: 044 8789022

Promega Biotech AB

Mikael Arnfelt M. Sc.
Sales Manager Norway, Sweden
E: mikael.arnfelt@promega.com
P: +46 705 65 65 79

Promega Biotech AB

Katja Ahokas
Sales Manager Finland, Estonia
E: katja.ahokas@promega.com
P: +358 45 615 8515