HDAC1 (human), (recombinant) (His-tag)

Manuals And Inserts

• Datasheet

Specific Protocol

Assay of HDAC1 (Prod. No. BML-SE456) with FLUOR DE LYS^®-SIRT1 substrate (Prod. No. BML-KI177) & FLUOR DE LYS^® Developer II (Prod. No. BML-KI176)

Components of Assay:
HDAC Assay Buffer II (Prod. No. BML-KI422)*
(50 mM Tris/Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/ml BSA (fatty acid free BSA, e.g. Sigma catalog # A3803))
HDAC1 (Prod. No. BML-SE456)*
Dilute to enough HDAC1 to 0.1 µg/µl with HDAC Assay Buffer II (Prod. No. BML-KI422) to provide 5 µl per well. Keep on ice until use.
FLUOR DE LYS^®-SIRT1 substrate (Prod. No. BML-KI177)*
Thaw quickly and keep on ice. Dilute 5 mM stock in HDAC Assay Buffer II.
2x Substrate Solution
Prepare a 2x substrate solution by diluting the 5 mM FLUOR DE LYS^®-SIRT1 substrate (Prod. No. BML-KI177) in HDAC Assay Buffer II (BML-KI422). Each assay well will require 25 µl (see below). For example, prepare 1 ml of 20 µM substrate (for final 10 µM) by mixing 4 µl of 5 mM FLUOR DE LYS^®-SIRT1 substrate (Prod. No. BML-KI177) and 996 µl HDAC Assay Buffer II (Prod. No. BML-KI422). Warm to 37°C before use. (NOTE: HDAC1's Km for FLUOR DE LYS- SIRT1 substrate (Prod. No. BML-KI177) is ~20 µM. Therefore, a reasonable substrate concentration for inhibitor screening would be 10-20 µM FLUOR DE LYS^®-SIRT1 (Prod. No. BML-KI177), while 100 or 200 µM would be more suitable for a specific activity measurement at a saturating substrate concentration.)
Trichostatin A (Prod. No. BML-GR309; HDAC Inhibitor)
Prepare a 0.2 mM stock in dimethylsulfoxide (DMSO). DMSO stock may be stored at -20°C. The 0.2 mM stock will be diluted 100-fold in 1x Developer II in order to stop HDAC activity at the start of the signal development process. To prepare a stock for use in testing trichostatin inhibition (i.e. for addition to the deacetylation phase of the reaction), dilute 0.2 mM stock to 10 µM in HDAC
Assay Buffer II (e.g. 5 µl plus 95 µl) and keep on ice. Addition of 2.5 µl of this 10 µM stock per well will result in strong inhibition (final [trichostatin A] = 500 nM)
FLUOR DE LYS^® Developer II (5x Concentrate, Prod. No. BML-KI176)*
Shortly before use, dilute 5x stock solution to 1x plus 2 μM trichostatin A. For example, prepare 1 ml by mixing 200 µl of the 5x Concentrate, 790 µl HDAC Assay Buffer II (BML-KI422) and 10 µl 0.2 mM trichostatin A in DMSO. Store the 1x Developer II plus trichostatin on ice until use. Do not store excess, but prepare freshly as needed.
FLUOR DE LYS^® Deacetylated Standard (Prod. No. BML-KI142)*
Dilute the 10 mM stock in DMSO to 1 µM with HDAC Assay Buffer II (Prod. No. BML-KI422).
½ Volume 96-well white micro-plate (BML-KI110)*
*Components of the HDAC1 FLUOR DE LYS^® Fluorescent Activity Assay (Prod. No. BML-AK511), which are also sold separately.

Reaction Condition Examples:

1. Designate wells for four reactions: 30 min rxn; 30 min rxn plus trichostatin A; 0 min rxn and a Standard well.
2. Add 20 µl of HDAC Assay Buffer II to the 30 min rxn well and the 0 min rxn well. To the third well (30 min. plus trichostatin) add 2.5 μl of 10 μM trichostatin A plus 17.5 µl of HDAC Assay Buffer II. Allow to equilibrate to assay temperature (37°C). (Leave Standard well empty until step 7).

<li<Add 5 µl of diluted HDAC1 (BML-SE456, 0.1 µg/µl) to the wells for the 0 min, 30 min, and 30 min. plus trichostatin rxns.

To start reactions, add 25 µl of the 2x Substrates (37°C) to both 30 min reaction wells. Allow reactions to run 30 min at 37C.

3. Add 50 µl of 1x Developer II plus trichostatin A to both 30 min. reaction wells.
4. To the 0 min rxn well, add 50 µl of 1x Developer II plus trichostatin A, immediately followed by 25 µl of 2x substrate.
5. For the standard, mix 50 µl of 1 μM standard with 50 µl of 1x Developer II plus trichostatin A in the fourth well.
6. Allow 45 min. at 37°C for signal to develop and then read plate in a microplate-reading fluorimeter capable of excitation at a wavelength in the range of 350-380 and detection of emitted light in the range of 440-460 nm.
7. Data analysis: Determine the ΔAFU (Arbitrary Fluorescence Units) for the two 30 min rxns. (AFU of 30 min rxn. (with or without trichostatin) minus AFU of the 0 min rxn). Determine AFU/pmol by dividing the Deacetylated standard reading (AFU) by 50 pmol. Calculate pmol of substrate deacetylated in 30 min (divide ΔAFU by AFU/pmol).

Certificate of Analysis

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