« Back
Detection of JumonjiC Domain-Containing Histone Demethylase activities With a Homogeneous Bioluminescent Succinate Assay
Detection of JumonjiC Domain-Containing Histone Demethylase activities With a Homogeneous Bioluminescent Succinate Assay
Submitted on 15 May 2017

Hicham Zegzouti, Juliano Alves, and Said Goueli
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711
Poster Views: 656
View poster »
Poster Abstract
JumonjiC domain containing histone lysine demethylases (JMJCs) play a pivotal role in determining the epigenetic status of the genome, leading to either transcriptional repression or activation of target genes by counteracting the activities of histone lysine methyltransferases. Because of their implication in cancer, they have become validated drug targets. Therefore, assays for this enzyme subfamily are desirable in order to facilitate the identification of selective and potent inhibitors for drug discovery and as basic research tools. Since succinate is one of the products of the demethylation reaction catalyzed by these enzymes, an assay that detects succinate would be suitable for monitoring all JMJC activities as well as other succinate-forming enzymes (i.e.: dioxygenases). Thus, a bioluminescent and homogenous succinate detection assay for measuring JMJC activity was developed, and it is performed in a two-step format that relies on converting the succinate product to ATP, and then to light in a robust luciferase reaction. The light output is proportional to succinate concentration from low nM to 15μM, and the assay is highly sensitive and robust, two features that are highly desirable and essential for measuring the activity of the majority of JMJC demethylase subfamilies. Therefore, the succinate detection assay is a simple-to-use method that does not require antibodies or modified substrates. Examples of various applications of this succinate detection assay will be presented, including studies on specificity of different substrates by diverse JMJCs, as well as mode of action studies using specific inhibitors. The development of this succinate detection assay will make it possible to investigate a large number of JMJC demethylases and could have significant impact on diverse areas of Epigenetics research.

Report abuse »
Ask the author a question about this poster.
Ask a Question »

Creative Commons

Related Posters

A Novel Enzymatic Assay for Determination of Phosphatidylinositol in Biological Samples
Paul Templeton, Kyle C. Schmitt, Grigoriy Tchaga,and Gordon Yan

A Chemically-Defined Baculovirus-Based Expression System for Enhanced Protein Production in Sf9 Cells
Maya Yovcheva, Sara Barnes, Kenneth Thompson, Melissa Cross, Katy Irvin, Mintu Desai, Natasha Lucki, Henry Chiou, Jonathan Zmuda

Development of an in vitro Model System for Newcastle Disease Virus Persistence in Bladder Cancer Cells
Ahmad U1,, Chan SC2, Chau DM1, Chia SL5, Abdullah S1,3, Yusoff K5 & Veerakumarasivam A1,4*

Salamanca Grosso, G.; Osorio Tangafarife, M.P.

The EurOPDX EDIReX project: towards a European Research Infrastructure on patient-derived cancer models
E. Vinolo 1, J.P. Morris 1, D.G. Alférez 2, J. Arribas 3,4,5, C. Bernadó 3,4,5, A. Bertotti 6, A. Bruna 7, A.T. Byrne 8, C. Caldas 7, R.B. Clarke 2, N. Conte 9, R. Corsi 10, S. Corso 6, M. Crespo 3, A. Dahmani 11, V. Dangles-Marie 11, D. Decaudin 11, Z. Dudová 12, A. Fiori 6, S. Giordano 6, M. Hauptsmann 13, M. Hidalgo 14, C. Isella 6, S. de Jong 15, J. Jonkers 13, A. Křenek 12, O. Krijgsman 13, D. Kouřil 12, J.C. Lacal 14, L. Lanfrancone 16, E. Leucci 17, G.M. Mælandsmo 18, E. Marangoni 11, J. Mason 9, M.Th. Mayrhofer 19, A. Mazzocca 6, T.S. Meehan 9, E. Montaudon 11, F. Nem