HDAC-Glo™ & SIRT-Glo™ Assays

Simple, fast and highly sensitive bioluminescent HDAC assays.

• Simple- add-mix-measure protocol
• Sensitive- 10-100 fold more sensitive than fluorescent substrate assays
• Fast – achieve maximal sensitivity in 15-45 minutes
• Scalable and Automatable- 96- to 1536-well plate formats
• Multiplex Compatible- HDAC-Glo™ Assay multiplexed with a viability assay
• Easy Counter Confirmation- Easy-to-use non-acetylated control substrates available to confirm HDAC inhibition

Assay Principal

The HDAC-Glo™ and SIRT-Glo™ Assays provide a single-reagent addition bioluminescent assay for easy, fast and sensitive measurement of HDAC activity.

All three enzymatic events occur in coupled, nearly simultaneous reactions upon addition of a single reagent and in 15-45 minutes with a signal half-life >3 hours. HDAC activity deacetylates the proluminogenic peptide substrate making it sensitive to proteolytic cleavage by a developer enzyme and liberates free luciferin which in the presence of Ultra-Glo™ rLuciferase and ATP produces light. The amount of light output correlates with HDAC activity.

Assay Protocol

Simple and Fast, Add-Mix-Measure Protocol

HDAC-Glo™ I/II Assay is broadly useful for enzymes in cells, from extracts and purified enzyme sources. The SIRT-Glo™ Assay is broadly useful for purified enzymes.

Sensitivity

10- to 100-fold higher sensitivity than comparable fluorescent assays reduces amount of enzyme required. Less interference minimizes false +/- results.

Broad, Linear Isoenzyme Utility

Automation Compatible

The assays produce large signal windows with excellent Z’ values in a simple add-mix measure format making them easily scalable and automatable for 96- to 384- well formats.

HDAC-Glo I/II Assay

 

SIRT-Glo Assay

Applications

The HDAC-Glo™ and SIRT-Glo™ Assays can be used for many screening applications, including: rank order potency profiling, defining differential inhibition, and counter-screening for selectivity. The HDAC-Glo™ Assay can be used in a cell-based multiplex format with the CellTiter-Fluor™ Cell Viability Assay to get more biologically relevant data per well.

Scaffold Inhibitor Profiling in a Multiplexed Format with the HDAC-Glo I/II Assay.

 

HDAC inhibition was monitored in conjunction with cell viability using U937 Cancer cells with different inhibitor classes, A: Hydroximates; B: Short-chain fatty acids. Resulting data were consistent with published IC₅₀ values.

Applications

Assay for Off-Target Effects in Primary Cells with the HDAC-Glo I/II Assay.

SAHA was applied to iPS derived Cardiomyocytes (iCell™ Cardiomyocytes from Cellular Dynamics International) for 24 hours. HDAC inhibition with the HDAC-Glo™ I/II Assay and cell viability with the CellTiter-Fluor™ Assay were determined. Potent HDAC inhibition was observed with no apparent cellular toxicity.

Applications

Reproducible IC₅₀ determination with the SIRT-Glo Assay.

Reproducible IC₅₀s were obtained with known sirtuin inhibitors with different mechanisms of action. The IC₅₀s obtained were comparable to those obtained using Fluor-de-Lys® Assays. The data shown is with rSIRT2.