HDAC Colorimetric Assay Kit

(Catalog #K331-100; 100 assays; Store kit at –20^oC; Lot #: 40431)

I. Introduction:

Inhibition of histone deacetylases (HDACs) has been implicated to modulate transcription and to induce apoptosis or differentiation in cancer cells. However, screening HDAC inhibitory compounds has proven to be difficult over the past due to the lack of convenient tools for analyzing HDAC activity. The new Colorimetric HDAC Activity Assay Kit provides a fast and convenient colorimetric method that eliminates radioactivity, extractions, or chromatography, as used in the traditional assays. The new method requires only two easy steps, both performed on the same microtiter plate. First, the HDAC colorimetric substrate, which comprises an acetylated lysine side chain, is incubated with a sample containing HDAC activity (e.g., HeLa nuclear extract or your own samples). Deacetylation of the substrate sensitizes the substrate, so that, in the second step, treatment with the Lysine Developer produces a chromophore. The chromophore can be easily analyzed using an ELISA plate reader or spectrophotometer. The assay is well suited for high throughput screening applications. HDAC inhibitors and antibodies are also available separately

1. 
   

   Kit Contents:

   
   

 

 

 

 

 

III. HDAC Assay Protocol:

A. General Consideration:

• 
  

  Read the entire protocol before beginning the procedure.

  
  


• 
  

  The HeLa nuclear extract and Lysine Developer should be refreeze immediately at –20 or -70^oC after each use to avoid loss of activity.

  
  


• 
  

  If positive and negative controls are designed, the kit provides sufficient reagents for 5 positive control assays with the HeLa Nuclear Extract and 5 Negative Control assays with the HDAC Inhibitor, Trichostatin A.

  
  


• 
  

  Using 96-well plates with U-shape bottom. Flat bottom may give a little low value.

  
  

B. Assay Protocol:

1. 
   

   Dilute test samples (50-200 g of nuclear extract or cell lysate) to 85 l (final volume) of ddH₂0 in each well (For background reading, add 85 l ddH₂0 only). For positive control, dilute 10 l of HeLa nuclear extract with 75 l ddH₂0. For negative control, dilute yoursample into 83 l of ddH₂0 and then add 2 l of Trichostatin, or use a known sample containing no HDAC activity.

   
   

 

 

 

2. 
   

   Add 10 l of the 10X HDAC Assay Buffer to each well.

   
   


3. 
   

   Add 5 l of the HDAC colorimetric substrate to each well. Mix thoroughly.

   
   


4. 
   

   Incubate plates at 37^oC for 1 hour (or longer if desired).

   
   


5. 
   

   Stop the reaction by adding 10 l of Lysine Developer and mix well. Incubate the plate at 37^oC for 30 min.

   
   


6. 
   

   Read sample in an ELISA plate reader at 400 or 405 nm. Signal is stable for several hours at room temperature. HDAC activity can be expressed as the relative O.D. value per g protein sample.

   
   

C. Standard Curve (optional):

1. 
   

   If desired, a standard curve can be prepared using the known amount of the Deacetylated Standard included in the kit. The exact concentration range of the Deacetylase Standard will vary depending on the each individual plate reader and the exact wavelength used. We recommend starting with a dilution range of 10-100 M in Assay Buffer.

   
   


2. 
   

   Add 90 l each of the dilutions and also 10 l of the 10X Assay Buffer into a set of wells on the microtiter plate. Use 90 l of H₂O and 10 l of 10X Assay Buffer as zero

   
   


3. 
   

   Add 10 l of Lysine Developer to each well and incubate at 37^oC for 30 min (Note: Incubation time should be kept the same for both standard and test samples.)

   
   


4. 
   

   Read samples in an ELISA plate reader at 400 or 405 nm.

   
   


5. 
   

   Plot O.D. value (y-axis) versus concentration of the Deacetylated Standard (x-axis). Determine the slope as O.D./M.

   
   


6. 
   

   Based on the slope, you can determine the absolute amount of deacetylated lysine generated in your sample.