A fluorogenic, acetylated peptide substrate for class2a HDACs (HDACs 4, 5, 7 and 9). It also works well for HDACs 8 and 11. This substrate exhibits longer excitation and emission wavelengths (excitation 485 nm, emission 528 nm) than our regular class 2a HDAC substrate, #50040, which fluoresces at excitation 350-380 nm, emission at 440-460 nm. This substrate should be used when the inhibitor fluoresces near 450 nm. If autofluorescence is not an issue, we recommend product #50040. For autofluorescent samples using HDACs 1-3 ,6, and 10, we recommend substrate #50038.

5 mM DMSO solution (50 µl)

DMSO solution

Materials :
HDAC Assay buffer (BPS catalog #50031)
HDAC Assay developer (BPS catalog #50030)
HDAC Class 2A Substrate 2 (green) (catalog #50042)
HDAC4 (0.2 ng/µl) (BPS catalog #50004) or other class2a HDAC enzyme

Step 1 :
Add the following reagents to a low binding NUNC black plate (VWR catalog #62408-936):
HDAC Assay Buffer, 35 µl
1 mg/ml BSA, 5 µl
200 µM HDAC 2A substrate 2, 5 µl
HDAC4, 5 µl
Incubate at 37°C for 30 min.

Step 2 :
Stop the reaction by adding 50 µl of HDAC assay developer (undiluted) and incubate the plate at room temperature for 15 min.

Step 3 :
Read samples in a microtiter-plate reading fluorimeter capable of excitation at 485 nm and emission at 528 nm. “Blank” value (no enzyme negative control) is subtracted from all other values.

Stable at least 12 months from date of receipt, when stored as directed

Study enzyme kinetics, and screen small molecule inhibitors of HDAC class2a for drug discovery and HTS applications.

1. A. Ito et al. (2001) EMBO J. 20 1331.
2. A. Ito et al. (2002) EMBO J. 21 6236.
3. N.A. Barlev et al. (2001) Mol. Cell 8 1243.

Protect from direct exposure to light. Avoid freeze/thaw cycles.

Deacetylase

Substrate