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European Tour

16 - 27 April 2018

Explore Innovation at the Molecular Level

New luminescent technologies are simplifying protein tagging and enabling protein detection in live cells without overexpression. Attend one of the 9 symposiums across Europe in April 2018 to discover a universe of possibilities.

Jolanta Vidugiriene, Ph.D.

Jolanta Vidugiriene, Ph.D.

Senior Scientist at Promega Corporation

Jolanta Vidugiriene is a Group Leader in R&D at Promega leading development of new technologies for mechanistic studies of cell survival and adaptation, particularly those involved in regulation of cellular energy metabolism.

Prior to joining Promega, Jolanta was a Principal Investigator of a Howard Hughes International Grant, where she conducted collaborative research between the Department of Biochemistry, University of Wisconsin-Madison and Vilnius University, Lithuania. Jolanta received her Ph.D. in biochemistry from Vilnius University, completed post-doctoral studies at Boston Biomedical Research Institute and Laboratory of Molecular Parasitology, Rockefeller University.

Topics & Abstracts

Intracellular signaling and cell function are largely mediated through protein dynamics, including changes in protein abundance, interactions, location, or post-translational modification. HiBiT is an 11-amino-acid quantitative peptide tag that is well suited for such studies. High-affinity complementation of HiBiT with the 18kDa polypeptide, known as large BiT (LgBiT), generates a bright luminescent enzyme, enabling sensitive and linear quantitation of HiBiT-tagged proteins over seven orders of magnitude. Changes in protein abundance are monitored in either a lytic endpoint assay using purified LgBiT protein or in a live-cell kinetic assay by expressing the LgBiT subunit in cells. HiBiT can also be used to measure translocation of proteins to the cell surface in a live-cell assay as LgBiT is membrane impermeable. Additionally, HiBiT-tagged proteins can be detected at sub-picogram levels on membranes following SDS-PAGE and transfer, using a simple protocol that eliminates multiple blocking, binding, and washing steps of traditional blotting techniques.

In this presentation we demonstrate applications of HiBiT technology for studying diverse biological responses including time-dependent protein secretion, PROTAC-induced degradation and insulin-induced translocation of proteins to the cell surface. We also show how the small size and brightness of HiBiT tag facilitates rapid knock-in at endogenous loci using a plasmid-free protocol with Cas9:gRNA ribonucleoprotein complex and a single-stranded oligonucleotide donor DNA, and demonstrate the advantages of studying protein dynamics under endogenous expression conditions as opposed to more common over-expression methods.

Better understanding the role of cell metabolism in cancer, immunology, obesity, diabetes, and neurodegenerative disease presents specific research challenges, which drive the need for more rapid and reliable methods for measuring changes in metabolic pathways. Metabolites produced by the major metabolic pathways serve as signaling molecules and link the metabolic state of a cell to transcriptional control, epigenetics and cell signaling. Therefore, the ability to measure changes in key metabolites rapidly and robustly in higher throughput formats (e.g., 96-, 384-well plates) should provide a powerful approach for establishing the links between cellular metabolism and cell function under normal and disease conditions.

Here we introduce a series of luminescent metabolite detection assays (i.e., for glucose uptake, glucose, lactate, glutamate, glutamine, and NAD(P)/NAD(P)H detection). We discuss how the assays provide valuable information about metabolic reprograming of cancer cells, allow monitoring the activation of T cells or measuring insulin sensitivity in primary adipocytes and muscle cells.

Continuous or 'real-time' monitoring of cellular processes provides a comprehensive representation of the changes occurring in live cells over time that informs on the biological status of the cell and enables decision making about the timing of treatments or the use of other functional end-point assays.

Here we introduce a new generation of sensitive, microtiter plate-based, live-cell assays that allow the continuous measurement of different biomarkers of cell health and apoptosis over several days. RealTime-Glo™ Cell Viability Assay uses the reducing potential of the cell as a measure of cell health. The assay is performed by adding NanoLuc® enzyme and a cell-permeable pro-substrate for NanoLuc at the time of cell plating or treatment. The bioluminescent signal that is generated directly correlates with the number of live cells and decreases rapidly upon cell death to allow the measurement of changes in real time. Bioluminescent Lactate Dehydrogenase (LDH) toxicity assay provides a simple, reliable method for quantifying LDH release into the culture medium. The assay sensitivity (LOD < 10 lysed cells) allows measurement of LDH release in a small amount of sampled medium and is easily adaptable for monitoring LDH release at different time points. The RealTime-Glo™ Annexin V Apoptosis Assay represents the first homogeneous reagent that allows real-time measurement in live cells of annexin V binding to exposed phosphatidylserine (PS), a well-established hallmark of the apoptotic phenotype.

The sensitivity and nondestructive nature of these new assays allow one to study time-dependent toxicity (of small molecule and biologics) in 2D and 3D cell models and can be multiplexed with other meaningful orthogonal cell health measures to confirm putative mechanisms of action. The resulting data enable you to extract previously hidden biology that may be missed when using only end point assays of viability, toxicity and apoptosis.

Promega United Kingdom

Ann Brine
M: 07384 461734
P: 023 8071 7319

Promega Benelux

Marylène Focant
M: +32 476 57 20 28
P: +31715324244

Promega Switzerland

Réka Nagy
P: 044 8789022

Promega Biotech AB

Mikael Arnfelt M. Sc.
Sales Manager Norway, Sweden
P: +46 705 65 65 79