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November 01, 2011 (Vol. 31, No. 19)

Detection of Lysine Deacetylase Activities

Bioluminescent Assays Offer Distinct Advantages over Fluorescent Approaches

  • Assay Sensitivity and Ease of Use

    Click Image To Enlarge +
    Figure 2. Luminescent and fluorescent assay sensitivity: HeLa nuclear extract or recombinant Sirt 2 enzyme (Enzo) assays were titrated in a 96-well plate. For HDAC-Glo I/II and SIRT-Glo Assays, Substrate Reagent was added to the deacetylase source, incubated for 15 minutes at room temperature, then data was measured. For fluorescent methods, fluorescent substrate was added to the deacetylase source and incubated at 37°C for 1 hour. Developer was then added for 15 minutes at room temperature, then data was measured. Data was plotted using GraphPad Prism® software. Data shown here represents the mean ± standard deviation of three samples.

    To benchmark assay sensitivity, HDAC-Glo I/II and SIRT-Glo Assays were compared to commercially available fluorescent assay methods (Figure 2). HeLa cell nuclear extracts or recombinant sirtuin 2 were titrated and assayed per the manufacturer’s instructions.

    The HDAC-Glo I/II and SIRT-Glo Assays delivered a 400-fold increase in sensitivity when compared to the fluorescent methods. This enhanced sensitivity allows for tangible cost savings due to a reduced requirement for recombinant enzyme and greater resolution of nonabundant deacetylase activities.

    In addition, there was a notable time savings associated with use of the HDAC-Glo I/II and SIRT-Glo Assays due to the relatively short 15-minute time period between reagent addition and data acquisition. In contrast, available fluorescent-based assays typically require multiple steps and more than two hours to complete.

  • High-Throughput Screening and Facile Detection of Assay Interferences

    All assays are subject to potential detection interferences that may generate false hits during high-throughput screening activities. To address possible assay interferences against the developer and luciferase enzymes, cognate, non-acetylated forms of the substrates were produced for counterscreening high-throughput screening hits against the coupled detection enzymes.

    These nonacetylated peptides can be introduced directly into the prepared reagent and contacted with the test compounds to determine a compound’s HDAC selectivity profile or reveal assay interferences. Halley et al., has successfully implemented the HDAC-Glo I/II, SIRT-Glo and counterscreen assays in a 1,536-well format and reported acceptable hit and false hit rates against the FDA 640 and Hypha Discovery MycoDiverse libraries.

  • Cell-Based HDAC Assays

    Click Image To Enlarge +
    Figure 3. Cell-based HDAC activity assays: HDAC inhibitors were applied to human embryonic stem cells for a period of one hour. HDAC-Glo I/II Reagent was added in a nonlytic format and HDAC activity measured.

    Cell-based HDAC experiments are especially relevant because they address the natural context of HDAC enzymes and can reflect the diversity of expression arising from different cell types. Furthermore, on-target HDAC potency can be linked to functional outcomes such as on- and off-target toxicities. Because the HDAC-Glo I/II Substrate is cell permeable, the assay can be conducted in either a lytic (with the addition of detergent) or nonlytic manner. Nonlytic formats are particularly attractive when the cell source is semi-precious and too difficult to obtain for parallel experimentation.

    As illustrated in Figure 3, human embryonic stem cells (hESC) were contacted with various HDAC inhibitors for a period of one hour. The HDAC-Glo I/II Reagent was then applied in a nonlytic formulation and HDAC activity measured. Medium and reagent could then be removed for additional experimentation such as RT-PCR.

    The hESC contained robust HDAC activity that can be inhibited in a dose-dependent manner and compounds rank ordered. The SIRT-Glo Substrate, while also cell permeable, is not recommended for cell-based applications due to HDAC cross-reactivity with the SIRT-Glo peptide sequence.

  • Summary

    The HDAC-Glo I/II and SIRT-Glo Assays provide users with a simple, homogeneous, “add-mix-measure” procedure for the specific detection of lysine deacetylase activities. These assays generate a robust, proportional, and persistent signal that remains stable for several hours, making them useful for basic research through high-throughput applications. Both assays are broadly responsive to recombinant enzymes, with the HDAC-Glo I/II Assay providing additional functionality for use in cell-based applications. The bioluminescent readout also confers distinct advantages over alternative fluorescent approaches, including higher sensitivity, shorter incubation times, and less complicated assay procedures. Moreover, data acquisition is faster than with fluorescent methods. HDAC-Glo I/II and SIRT-Glo Assays also offer a counterscreening method with nonacetylated substrate for high-throughput activities that detect assay interferences.

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