Amplite® Fluorimetric HDAC Activity Assay Kit Green Fluorescence

Histone deacetylases (HDAC) are a class of enzymes that remove acetyl groups from a ε-N-acetyl lysine amino acid on a histone. Deacetylation restores the positive electric charge of the lysine amino acids, which increases the histone's affinity for the negatively charged phosphate backbone of DNA. This generally down-regulates DNA transcription by blocking the access of transcription factors. HDACs are involved in the pathway by which the retinoblastoma protein (pRb) suppresses cell proliferation. The pRb protein is part of a complex which attracts HDACs to the chromatin so that it will deacetylate histones. HDAC inhibitors are being studied as a treatment for cancer. The Amplite® HDAC Assay Kit provides a quick, convenient, and sensitive method for the detection of HDAC activity. Our HDAC Green™ substrate is a non-peptide compound that is much more resistant than other commercial peptide-based HDAC substrates. Our kit can be used for measuring HDAC activity in cell lysates, in vitro inhibitor screening with extracts or purified enzymes. The long wavelength emission and higher extinction coefficient of the HDAC Green™ substrate provide less interference from compounds and cell components. HDAC activity is determined by monitoring the green fluorescence enhancement with excitation at 490 nm and emission at 520 nm.

Example protocol

AT A GLANCE

Protocol Summary

Prepare HDAC containing samples (40 µL)
Add HDAC inhibitor or test compounds (10 µL)
Incubate at room temperature or 37°C for 10 - 20 minutes
Add HDAC Green™ Substrate working solution (50 µL)
Incubate at room temperature or 37°C for 30 - 60 minutes
Monitor fluorescence intensity at Ex/Em = 490/525 nm (Cutoff = 515 nm)

Important Thaw all the kit components before starting the experiment.

PREPARATION OF WORKING SOLUTION

1. HDAC-containing test samples

Dilute 5 - 10 mg/mL of HeLa nuclear extract or cell lysates at 1:40 in Assay Buffer (Component B).
Note 40 µL of the diluted sample is enough for one well of a 96-well plate. Dilute extract immediately before use. Store the solution on ice.

2. HDAC inhibitor (Trichostain A) solution (30 µM)

Dilute 3 mM Trichostatin A solution (Component C) at 1:100 in assay buffer (Component B) to get a 30 µM Trichostatin A solution. Add 10 μL of the 30 μM Trichostatin A solution into each inhibitor control well.

3. HDAC Green™ Substrate working solution

Add 20 μL of HDAC Green™ Substrate (Component A) and 100 μL of the Signal Enhancer (Component D) into 5 mL of Assay Buffer (Component B).
Note The diluted HDAC Green™ Substrate working solution is not stable. 5 mL of the diluted HDAC Green™ Substrate working solution is enough for 100 assays. Prepare fresh HDAC Green™ Substrate working solution for each experiment. Keep reconstituted working solution on ice until use.

SAMPLE EXPERIMENTAL PROTOCOL

Table 1. Layout of nuclear extracts with test compounds in a solid black 96-well microplate.

Samples	HeLa Extract	Assay Buffer (Component B)	Trichostatin A (HDAC inhibitor)	Test Compounds	HDAC Green™ Substrate
Blank (no enzyme)	0 µL	50 µL	0 µL	0 µL	50 µL
Positive Control	40 µL	10 µL	0 µL	0 µL	50 µL
Negative Control	40 µL	0 µL	10 µL	0 µL	50 µL
Test Compound	40 µL	0 µL	0 µL	10 µL	50 µL

Add 40 µL of diluted nuclear extract, enzyme solution or other HDAC samples, and 10 µL of test compounds to the corresponding microplate wells.
1. For positive control: Add 40 µL of diluted HDAC enzyme solution or HeLa nuclear extract with 10 µL of Assay Buffer (Component B).
2. For negative control: Add 40 µL of diluted HeLa nuclear extract with 10 µL of 30 µM Trichostatin A solution, or use a known sample containing no HDAC activity.
3. For Blank (no Enzyme): Add 50 µL of Assay Buffer (Component B) only.
Incubate the plate at room temperature or 37°C for 10 - 20 minutes.
Note For screening HDAC inhibitor, preincubate the compounds with HeLa nuclear extract or pure enzyme before adding HDAC Green™ Substrate working solution.
Add 50 µL of HDAC Green™ Substrate working solution into each well. Incubate the plate at room temperature or 37°C for 30 - 60 minutes.
Monitor fluorescence intensity at Ex/Em = 490/525 nm. (Cutoff = 515 nm)

Spectrum

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Citations

View all 19 citations: Citation Explorer

Electroacupuncture Promotes the Generation of Intestinal Treg Cells After Ischemic Stroke by Foxp3 Acetylation Regulation
Authors: Chen, Yonglin and Ouyang, Ling and Yang, Xinyi and Wu, Bufan and Meng, Lingling and Gu, Jialin and Wang, Yaling and Li, Juan and Zhang, Jingjing and Jing, Xinyue and others,
Journal: Molecular Neurobiology (2024): 1--15

Target-based drug discovery: Applications of fluorescence techniques in high throughput and fragment-based screening
Authors: Kumar, Vikrant and Lakshman, Puneeth Kumar Chunchagatta and Prasad, Thazhe Kootteri and Manjunath, Kavyashree and Bairy, Sneha and Vasu, Akshaya S and Ganavi, B and Jasti, Subbarao and Kamariah, Neelagandan
Journal: Heliyon (2023): e23864

Porcine deltacoronavirus infection cleaves HDAC2 to attenuate its antiviral activity
Authors: Li, Zhuang and Fang, Puxian and Duan, Panpan and Chen, Jiyao and Fang, Liurong and Xiao, Shaobo
Journal: Journal of Virology (2022): e01027--22

Influence of Dinitrosyl Iron Complexes on the Activity of Enzymes, Indicators of Cardiovascular Diseases
Authors: Akentieva, Natalia and Sanina, Natalia and Gizatullin, Artur and Shkondina, Natalia and Andreeva, Anna and Shram, Stanislav and Aldoshin, Sergei
Journal: (2022)

Histone deacetylase 5 deacetylates the phosphatase PP2A for positively regulating NF-$\kappa$B signaling
Authors: Xu, Chonghui and Tang, Jielin and Yang, Qi and Zhao, He and Liu, Yaling and Cao, Juan and Zhou, Yuan and Chen, Xinwen and Chen, Jizheng
Journal: Journal of Biological Chemistry (2021): 101380

References

View all 80 references: Citation Explorer

DNA damage promotes histone deacetylase 4 nuclear localization and repression of G2/M promoters, via p53 C-terminal lysines
Authors: Basile V, Mantovani R, Imbriano C.
Journal: J Biol Chem (2006): 2347

The histone deacetylase inhibitor trichostatin a has genotoxic effects in human lymphoblasts in vitro
Authors: Olaharski AJ, Ji Z, Woo JY, Lim S, Hubbard AE, Zhang L, Smith MT.
Journal: Toxicol Sci (2006): 341

Histone deacetylase (HDAC) 4 involvement in both Lewy and Marinesco bodies
Authors: Takahashi-Fujigasaki J, Fujigasaki H.
Journal: Neuropathol Appl Neurobiol (2006): 562

Experimental therapy of malignant gliomas using the inhibitor of histone deacetylase MS-275
Authors: Eyupoglu IY, Hahnen E, Trankle C, Savaskan NE, Siebzehnrubl FA, Buslei R, Lemke D, Wick W, Fahlbusch R, Blumcke I.
Journal: Mol Cancer Ther (2006): 1248

Role of histone deacetylase Rpd3 in regulating rRNA gene transcription and nucleolar structure in yeast
Authors: Oakes ML, Siddiqi I, French SL, Vu L, Sato M, Aris JP, Beyer AL, Nomura M.
Journal: Mol Cell Biol (2006): 3889

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