HDAC activity assay - (Dec/05/2008 )

If you already made such experiment so what kind of kit did you use?Which company?Fluorometric or colourimetric modification?

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Hey Roman80

in our lab we used HDAC Fluor De Lys fluorescent activity assay from BIOMOL to determine IC50 of some HDAC-i.
Here is the protocol;
IC50 determination was performed using HDAC Fluor De Lys Fluorescent activity assay/drug discovery kits from BIOMOL. Briefly, cellular proteins were extracted by sonification of hepatocytes in buffer containing 50mM TrisHCl, 0.25mM sucrose, 25mM KCl and 5mM MgCl2. After centrifugation (5 min, 16000g) the protein concentration in the supernatant was determined according to Bradford, using BSA as a standard. Stock solutions of HDACi in pure ethanol were serially diluted in assay buffer containing 25mM Tris HCl, 137mM NaCl, 1mM MgCl2 x 6H2O. 10µl was added to 96-well microplates, followed by 15µl of cellular protein extract and 25µl Fluor de Lys substrate. After 15 minutes, the reaction was stopped by addition of 50µl developer. After another 15 minutes, the results were read in a microplate reading fluorimeter (Victor 3, Perkins Elmer) at 460nm against appropriate controls.
greetz

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