Table 3. General reagent scheme for the fluorometric HDAC activity assay.

HCT116 nuclear extraction (high HDAC activity) was used as a positive tissue control. Volume of HCT116 needed depends on protein content calculated from BCA assay; keep in mind that protein content differs between tissue type. Abbreviation: Ac, acetylated substrate.

Sample	Assay buffer	Inhibitor TSA (5×)	Tissue Extract (minimum 3 μg/15μl)	Substrate (Ac, 100 μM)		Diluted Developer	Total (μl)
Blank No enzyme	25 μl	—	—	25 μl	Equilibrate buffer, HCT116 extract and inhibitors to assay temperature. Initiate HDAC reaction by adding substrate, Incubate 30 min at 37°C. Add developer (15 min) to stop reaction	50 μl	100
Tissue sample	10 μl	—	15 μl	25 μl		50 μl	100
Tissue + Trichostatin A (0.1 μM stock)	—	10 μl	15 μl	25 μl		50 μl	100
Nuclear extract from HCT116 cells, if needed	23		2 μl	25 μl		50 μl	100
Nuclear extract from HCT116 cells +TSA, if needed	13	10 μl	2 μl	25 μl		50 μl	100
Deacetylated Standard (BL, if need. see below)	varies	Various amounts of standard needed to have the final concentrations of standard at 0 μM - 2.5 μM in 50 μl of assay buffer				50 μl	100

Concentration of test inhibitors depends on their potency (IC₅₀ for HDACi).