A fluorogenic, acetylated peptide substrate for HDACs. It is a suitable substrate for class I HDACs (HDACs 1, 2, and 3) and class 2b HDACs (HDACs 6 and 10). This substrate exhibits longer excitation and emission wavelengths (excitation 485 nm, emission 528 nm) than our regular class 1/2b HDAC substrate, #50037, 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 HDAC substrate 3, #50037. For autofluorescent samples using HDACs 4, 5, 7, 8, 9, and 11, we recommend substrate #50042.

5 mM DMSO solution (50 µl)

DMSO solution

Materials :
HDAC Assay buffer (BPS catalog #50031)
HDAC Assay developer (BPS catalog #50030)
HDAC Substrate 2 (green) (catalog #50038)
HDAC1 (0.33 µg/µl) (BPS catalog #50051) or other class1/2b HDAC enzyme

Step 1 :
Add the following reagents to a low binding NUNC black plate (VWR catalog #62408-936):
HDAC Assay Buffer, 38 µl
1 mg/ml BSA, 5 µl
200 µM HDAC substrate 2, 5 µl
HDAC1, 2 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 HDAC inhibitors 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.

Product Specific Reference(s):
1. Day, JA., et al.,  J Med Chem. 2013 Oct 24;56(20):7997-8007. 

Application Reference(s):

1. The first report of direct inhibitors that target the C-terminal MEEVD region on heat shock protein 90 (2016)

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

Deacetylase

Substrate