HDAC-Glo 2 Assay (10ml)

The HDAC-Glo™ Class IIa and HDAC-Glo™ 2 Assays are single-reagent-addition, homogeneous, luminescent assays that measure the relative activity of histone deacetylase (HDAC) Class IIa and Class I enzyme 2, respectively, 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 Luciferase (firefly). The signal from the assay reaction can be measured within 15–45 minutes after reagent addition with no sample manipulation. The HDAC-mediated luminescent signal is persistent, with a half-life of greater than 2 hours, allowing batch processing of multiwell plates.

Features - Benefits

• Provide Relevant Insight into Compound Effects in Biological Setting: Make better decisions about your compound library early in drug screening.
• Panel of Screening Tools Allows Comprehensive Screening of HDAC Activity: Easy detection of Class IIa or Isozyme 2 in the same, convenient platform.
• Highly Sensitive: Feel confident because you can see more. Obtain a dynamic range 10- to 100-fold higher than comparable fluorescence methods.
• Flexible Format: Determine inhibitor performance in both biochemical and predictive cell-based formats using viable cells or in vitro with cell extracts or purified recombinant enzymes.
• Simple Measurement of Deacetylating Activities: Easy implementation from benchtop to screening with a single-reagent-addition, homogeneous, add-mix-measure protocol.
• Fast Data Acquisition in as Little as 15 Minutes: 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.
• Robust Detection: Minimize assay interference often encountered with fluorescent assays with robust, bioluminescence-based detection. This technology also allows you to multiplex with cell-health assays, offering more biologically relevant data within a predictive, cell-based context.

Applications

• Determine HDAC inhibitor potency using purified enzymes, extracts or cells directly in culture plates.
• Selectively profile HDAC inhibitors with purified enzymes.
• Correlate HDAC inhibitor potency with cellular fate in same-well multiplexed viability assays.
• Determine off-target HDAC effects of compounds.

Notes

Cat.# G9560 and G9590 are sufficient to perform 100 × 100µl assays in 96-well plates or 1,000 × 10µl biochemical-based assays in 384-well plates (500 × 20µl cell-based assays in 384-well plates).