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DISCOVER-GLO: Bioluminescent Cell-Based Assay Seminar

Please join us to learn more about the latest developments and applications in luminescence cell-based assays. We will be featuring presentations on the novel applications of NanoLuc™ Luciferase for reporter assays and technologies for interrogating protein-protein interactions in living cells. Take the opportunity to network with fellow scientists and speakers during breaks and lunch.

  • Location: Creation Theatrette, Level 4 Matrix, Biopolis, Singapore. Location Map
  •  Monday, August 31, 2015
  •  12:00 PM–4:00 PM (SGT: Singapore)

SGworkshop@promega.com

Program Outline

12:00 pm  Arrival, Registration & Lunch / Networking
01:00 pm NanoLuc®: NextGen Luciferase for Analysis of Protein Dynamics and Cell Signaling 
Dr. Frank Fan, Director of Research
Promega Corporation
01:45 pm Assays for High Throughput Drug Screening
Dr. Choong Meng Ling, Group Leader, HTS
Experimental Therapeutics Centre (ETC)
02:15 pm Tea Break 
02:45 pm  Luciferase Based Assays for High Throughput Screening (HTS) in Infectious Diseases Drug Discovery
Dr. Srinivasa Rao, Investigator, Drug Discovery Unit
Novartis Institute for Tropical Disease (NITD)
03:15 pm NanoBRETTM and NanoBiTTM: New Tools for the Analysis of Protein Interactions in Living Cells
Dr. Frank Fan, Director of Research
Promega Corporation
04:00 pm Prize Draw / Tea Break

For more information on the seminar, download the program flyer here.


ABSTRACTS

NanoLuc®: NextGen Luciferase for Analysis of Protein Dynamics and Cell Signaling
Dr. Frank Fan, Promega Corporation

The growing need to study cell biology under physiological conditions with a single copy of the reporter in a single cell demands a brighter luciferase.  Promega's proprietary NanoLuc® (Nluc) luciferase, a small (19.1kDa) luminescent reporter, is >100-fold brighter than firefly or Renilla luciferases. The exceptional brightness of NanoLuc® combined with new genome editing technologies (via homologous recombination, TALEN or CRISPR) allows for the investigation of cellular events in the most physiologically-relevant conditions. When appended with a 41 amino-acid PEST sequence (P), the destabilized NlucP has a shortened intracellular half-life, therefore improving transcriptional dynamics and assay sensitivity.  NanoLuc® and firefly luciferase may be coupled together as a novel dual-reporter assay with improved sensitivity and dynamic range. Additionally, this combination (Nluc and Fluc) allows for the development of co-incidence reporter assays with lowered false hit rates compared to conventional dual-reporter assays. This is helpful in minimizing false positives especially in drug discovery high-throughput screening (HTS). Furthermore, we will show how NanoLuc® can be used for measure of protein dynamics, including changes in protein half-life and ligand-induced endocytosis of surface receptors. Also, NanoLuc® is the first luciferase that is amenable to real-time imaging of protein translocation events at high temporal and spatial resolution. This seminar will feature applications of NanoLuc® as a genetic reporter for cell based assays and imaging in both lytic and live-cell formats.

Download presentation slides here.

ChalkTalk video: "Why the Brightness of NanoLuc® Luciferase Matters?"

NanoBRET™ and NanoBiT™ : New Tools for the Analysis of Protein Interactions in Living Cells
Dr. Frank Fan, Promega Corporation

All aspects of protein function and activity are governed by an intricate network of interactions between proteins and other molecular constituents. The dynamic nature of these interactions and their physiological relevance can be revealed using analytical methods that do not disrupt the intracellular context. Here we present the development of two novel technology platforms, NanoBRET™ and NanoLuc® Binary Technology (NanoBiT™), that enable the investigation of protein interactions 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. When fused with a protein of interest (POI), NanoLuc® reports the function of the POI with regards to cellular localization, intracellular lifetime and most importantly interactions with macromolecules (i.e. protein, nucleic acid) and small molecules such as chemical drugs.  Protein:protein interaction (PPI) can be investigated with NanoBRET™ (NanoLuc® Bioluminescent Resonance Energy Transfer) and NanoBiT™ (NanoLuc® Binary Technology) technologies.  A variety of specific NanoBRET™ PPI assays were developed to aid drug discovery.  They include epigenetic proteins such as bromodomain, transcriptional proteins, signaling proteins, kinases, membrane proteins and RNA binding proteins. NanoBRET™ technology also enables investigation of protein:small molecule interactions such as engagement of small molecule drugs to its cellular protein target. NanoBiT™ technology is based on a PPI- facilitated structural complementation between a large fragment of NanoLuc® (156 amino acids) and a highly evolved 11 amino acids small peptide. It provides a simple system to study intracellular PPI in live cells.

Download presentation slides here.

ChalkTalk video: "Deciphering Biological Mysteries with NanoBRET™."

Luciferase Based Assays for High-Throughput Screening (HTS) in Infectious Diseases Drug Discovery
Dr. Srinivasa Rao, Investigator, Drug Discovery Unit

Luciferase based assays have been employed in identifying growth proliferation inhibitors in anti-bacterial and anti-parasitic drug discovery. Intracellular ATP levels are measured as surrogate markers for cell viability by providing external luciferin and luciferase. We developed whole cell based high through put screening assays for multiple infectious organisms such as Mycobacterium tuberculosis, M. bovis BCG, Trypanosoma brucei brucei and T. b. gambiense.  Luminescent Cell Viability Assay which determines metabolically active cells by ATP quantitation are sensitive, easy to operate, involves minimal handling and can generate large data rapidly. The homogeneous ‘add-mix-measure’ format causes cell lysis and generates a luminescent signal indicating the amount of ATP present in the culture. The assay makes use of the properties of a thermostable luciferase which generates a stable luminescent glow with prolonged half-life, an important advantage for compound library screens. Other advantages of the assay include high signal to background (S/B) ratio (useful for single point screens) and reproducibility. The luciferase based ATP determination, not only helps in identify growth inhibitors by HTS but also help in understanding basic biology of mycobacterial dormancy. Currently, we are attempting to utilize luciferase based assays for continuous monitoring of cell proliferation.

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