Fluorogenic HDAC Substrate 3

Catalog #: 50037
Lot#: 110124
Size: 250 nmole

DESCRIPTION: A fluorogenic, acetylated peptide substrate for HDACs. It is a suitable
substrate for many HDACs tested so far except class 2a HDAC.

PURITY: >95% by HPLC.

APPLICATIONS: Study enzyme kinetics, and screen small molecule inhibitors for drug
discovery and HTS applications.

SUPPLIED AS: DMSO solution

QUANTITY: 50 μl @ 5 mM

STORAGE: -20°C or -70°C.

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

Assay Protocol:

Material: Assay buffer (BPS #50031); Assay developer (BPS #50030); HDAC
Substrate (BPS #50037)

Step 1: Adding all reaction mixture to a low binding NUNC black plate (VWR
catalog number 62408-936)

2 ul of HDAC1 (0.33 ug/ul)
38 ul of HDAC assay buffer (BPS #50031)
5 ul of 1 mg/ml BSA
5 ul of 200 uM substrate (BPS #50037)
Incubate at 37 ºC for 30 min.

Step 2: Stop the reaction
Add 50 ul of HDAC assay developer (2x)(BPS #50030) and incubate the plate at
room temperature for 15 min

Step 3: Read sample in a microtiter-plate reading fluorimeter capable
of excitation at a wavelength in the range 350-380 nm and detection of
emitted light in the range 440-460 nm.