Amplite® Fluorimetric HDAC Activity Assay Kit *Green Fluorescence*

1. Prepare HDAC containing samples (40 µL)
2. Add HDAC inhibitor or test compounds (10 µL)
3. Incubate at room temperature or 37°C for 10 - 20 minutes
4. Add HDAC Green™ Substrate working solution (50 µL)
5. Incubate at room temperature or 37°C for 30 - 60 minutes
6. Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important Note

Thaw all the kit components before starting the experiment.

Dilute 5 - 10 mg/mL of HeLa nuclear extract or cell lysates at 1:40 in Assay Buffer (Component B).

Note: 40 µL of the diluted sample is enough for one well of a 96-well plate. Dilute extract immediately before use. Store the solution on ice.

Dilute 3 mM Trichostatin A solution (Component C) at 1:100 in assay buffer (Component B) to get a 30 µM Trichostatin A solution. Add 10 μL of the 30 μM Trichostatin A solution into each inhibitor control well.

Add 20 μL of HDAC Green™ Substrate (Component A) and 100 μL of the Signal Enhancer (Component D) into 5 mL of Assay Buffer (Component B).

Note: The diluted HDAC Green™ Substrate working solution is not stable. 5 mL of the diluted HDAC Green™ Substrate working solution is enough for 100 assays. Prepare fresh HDAC Green™ Substrate working solution for each experiment. Keep reconstituted working solution on ice until use.

Table 1. Layout of nuclear extracts with test compounds in a solid black 96-well microplate.

1. Add 40 µL of diluted nuclear extract, enzyme solution or other HDAC samples, and 10 µL of test compounds to the corresponding microplate wells.

   1. For positive control: Add 40 µL of diluted HDAC enzyme solution or HeLa nuclear extract with 10 µL of Assay Buffer (Component B).
   2. For negative control: Add 40 µL of diluted HeLa nuclear extract with 10 µL of 30 µM Trichostatin A solution, or use a known sample containing no HDAC activity.
   3. For Blank (no Enzyme): Add 50 µL of Assay Buffer (Component B) only.

2. Incubate the plate at room temperature or 37°C for 10 - 20 minutes.
   
   Note: For screening HDAC inhibitor, preincubate the compounds with HeLa nuclear extract or pure enzyme before adding HDAC Green™ Substrate working solution.

3. Add 50 µL of HDAC Green™ Substrate working solution into each well. Incubate the plate at room temperature or 37°C for 30 - 60 minutes.

4. Monitor fluorescence intensity at Ex/Em = 490/525 nm. (Cutoff = 515 nm)