HDAC-Glo™ I/II Assays and Screening Systems

The HDAC-Glo™ I/II Assays and Screening Systems are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. The assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is q...

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The HDAC-Glo™ I/II Assays and Screening Systems are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) class I and II enzymes from cells, extracts or purified enzyme sources. The assays use an acetylated, live-cell-permeant, luminogenic peptide substrate that can be deacetylated by HDAC activities. Deacetylation of the peptide aminoluciferin substrate is measured using a coupled enzymatic system in which a protease in the Developer Reagent cleaves the deacetylated peptide from the aminoluciferin substrate, releasing aminoluciferin, which is quantified in a reaction using Ultra-Glo™ recombinant firefly luciferase. The assay reaction is typically complete within 15–45 minutes with no sample manipulation. The HDAC-mediated luminescent signal is persistent, with a half-life of greater than 3 hours, allowing batch processing of multiwell plates. The HDAC assay is broadly useful for class I and II enzymes.

The Trichostatin A, included in the HDAC-Glo™ I/II Screening Systems or available separately, is a known pan HDAC inhibitor that may be used as a positive control inhibitor. The Trichostatin A is supplied at a concentration of 10mM in DMSO.

The HeLa Nuclear Extract, included in the HDAC-Glo™ I/II Screening Systems or available separately, may be used as a source of histone deacetylase activity. The diluted extract also can be used as an HDAC-Glo™ I/II Assay chemistry control.

The HDAC-Glo™ I/II Control Substrate, only available separately, is a non-acetylated form of the HDAC-Glo™ I/II Substrate with the same amino acid sequence and can be used with the HDAC-Glo™ I/II Assays and Screening Systems to confirm true HDAC inhibition in secondary screens. The Control Substrate is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plate format when combined with the HDAC-Glo™ Reagent prepared with components in the HDAC-Glo™ I/II Assays or Screening Systems. 

Features - Benefits

• Simple Measurement of Deacetylating Activities: Use a single-reagent-addition, homogeneous, add-mix-measure protocol for easy implementation from benchtop to screening.
• Highly Sensitive: Obtain 10- to 100-fold higher sensitivity than comparable fluorescence methods.
• Fast Data Acquisition: Achieve maximum signal in as little as 15 minutes with persistent glow-type steady-state signal, making the protocol amenable to automation in high-throughput formats and compatible with luminometers without injectors.
• Flexible to Sample Type: Use with viable cells, extracts or purified recombinant enzyme sources.

Applications

• Determine HDAC inhibitor potency using purified enzymes, extracts or cells directly in culture plates.
• Profile HDAC inhibitors with purified enzymes.
• Correlate HDAC inhibitor potency with cellular fate with same-well multiplexed viability assays.
• Confirm deacetylase inhibition with control substrates.

Notes

G6420 is sufficient for 100 assays at 100μl/assay in a 96-well plate or 400 assays at 25μl/assay in a 384-well plate. G6421 is sufficient for 500 assays at 100μl/assay in a 96-well plate or 2,000 assays at 25μl/assay in a 384-well plate. G6422 is sufficient for 1,000 assays at 100μl/assay in a 96-well plate or 4,000 assays at 25μl/assay in a 384-well plate.

G6430 is sufficient for 100 assays at 100μl/assay in a 96-well plate or 400 assays at 25μl/assay in a 384-well plate. G6431 is sufficient for 500 assays at 100μl/assay in a 96-well plate or 2,000 assays at 25μl/assay in a 384-well plate.

G6550 is supplied at a concentration of 10mM and is sufficient for 480 assays in 96-well plates when combined with the HDAC-Glo™ I/II Reagent.

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