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

BML-SE456-0050	50 µg		569.00 USD
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Product Details

Alternative Name:	Histone deacetylase 1
MW:	55 kDa
Source:	Produced in insect cells. HDAC1 from human cDNA (482 aa). Produced in a baculovirus expression system.
UniProt ID:	Q13547
Gene/Protein Identifier:	NM_004964 (RefSeq)
Formulation:	Liquid. In 50mM TRIS, pH 8.0, 138mM sodium chloride and 10% glycerol.
Purity Detail:	Partially purified by single-step affinity chromatography and gel filtration.
Specific Activity:	≥5 U/µg. One U=1 pmol/min at 37°C, 100µM, FLUOR DE LYS®-SIRT1 deacetylase substrate (Prod. No. BML-KI177).
Shipping:	Dry Ice
Long Term Storage:	-80°C
Use/Stability:	The enzyme is stable on ice for the time typically required to set up an experiment (30-60 min.), but may lose activity with prolonged storage on ice. It is recommended that thawing and dilution of the enzyme be done within as short a time as possible before start of the assay. The remaining, unused, undiluted enzyme should be refrozen quickly by, for example, snap freezing in a dry/ice ethanol bath or liquid nitrogen. Freezing and storage of diluted enzyme is not recommended.
Scientific Background:	Human HDAC1 (HD1) was the first protein to be linked to histone deacetylase activity. It is homologous to the yeast protein Rpd31, a relationship which has since come to define the “class I HDACs”. HDAC1 promotes transcriptional repression by deacetylating lysine ε-amino groups in histone N-terminal tails, a function frequently carried out in association with multi-protein transcription repression complexes such as NuRD3, Sin34 and CoREST6. Ubiquitously expressed in human tissues HDAC1-containing complexes appear to contribute the greater part of (at least class I) deacetylase activity in HeLa nuclear extracts. Aside from its interaction with co-repressors, HDAC1 activity may be regulated by post-translation modifications such as phosphorylation9 and sumoylation or binding to the inhibitor maspin, a tumor-suppressive serpin homolog. Although originally described as a “histone deacetylase”, HDAC1 has been shown to catalyze the regulatory deacetylation of non-histone proteins, including p53. Overexpression of HDAC1 has been found in various cancer types. HDAC inhibitors (HDACi) have shown considerable promise as anti-cancer agents and HDACi compounds from multiple chemical classes are in stages of drug development ranging from preclinical to phase III trials.
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: Designate wells for four reactions: 30 min rxn; 30 min rxn plus trichostatin A; 0 min rxn and a Standard well. 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). 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. Add 50 µl of 1x Developer II plus trichostatin A to both 30 min. reaction wells. To the 0 min rxn well, add 50 µl of 1x Developer II plus trichostatin A, immediately followed by 25 µl of 2x substrate. For the standard, mix 50 µl of 1 μM standard with 50 µl of 1x Developer II plus trichostatin A in the fourth well. 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. 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).
Regulatory Status:	RUO - Research Use Only

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