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Epigenetics & Chromatin

Histone Demethylase Assay (Fluorescent)

screen LSD1 enzymes for histone demethylase activity

The Histone Demethylase Assay (Fluorescent) provides a simple solution for screening activity of purified or recombinant lysine specific demethylase (LSD1, also known as KDM1) enzymes. The Recombinant Histone H3K4me2 substrate used in the assay mimics a native histone substrate, allowing for results that more closely resemble in vivo conditions. As the LSD1 enzyme demethylates the histone substrate, formaldehyde is released as a by-product. The Detection Reagent reacts with each formaldeyde molecule to generate a fluorescent signal equivalent to the overall production of formaldehyde. The fluorescent signal can be measured using a fluorescent microplate reader with an excitation wavelength of 410 nm and an emission wavelength of 480 nm.

For complete assay details, click the Method tab below. To learn more about lysine specific histone demethylase enzymes or to see assay data, click the LSD1 Info tab below; kit manuals can be downloaded under the Documents tab.

 
Name Format Cat No. Price  
Histone Demethylase Assay (Fluorescent) 48 rxns 53200 $365 Buy Now

Histone Demethylase LSD1 (KDM1)

Histone methyltransferases (HMTs) and histone demethylases (HDMs) regulate histone methylation. There are currently two classes of histone demethylase enzymes: the lysine specific demethylase 1 (LSD1) class and the Jumonji (JmjC)-domain-containing class. Each class of histone demethylase uses a different reaction mechanism, which leads to different substrate specificity and requires the use of different co-factors for enzymatic activity.

The lysine specific demethylase 1 (LSD1) class, also known as KDM1 for lysine (K) demethylase 1, uses a flavin adenine dinucleotide (FAD)-dependent amine oxidase domain for its demethylase activity. FAD is used as a cofactor to catalyze an amine oxidation that results in the release of a formaldehyde molecule. LSD1 can use either di- or mono-methylated H3K4 as a substrate. If a dimethyl H3K4 substrate is used, the demethylation reaction will produce a monomethyl H3K4 substrate that is then capable of undergoing a subsequent reaction to become unmethylated. A formaldehyde molecule will be released at each step.

Histone Demethylase LSD1 Activity

The Histone Demethylase Assay (Fluorescent) can be used to analyze the overall histone conversion efficiency of an LSD1 sample, or to screen compounds for changes in histone demethylation activity.

 

Histone demethylase LSD1 (KDM1) activity.
 
Figure 1: Fluorescence detection of LSD1 demethylase activity.

The Histone Demethylase Assay was used to assay activity of the LSD1 positive control enzyme. One µg of LSD1 was tested in the absence or presence of the recombinant H3K4me2 histone substrate. The enzymatic reaction was incubated at 37°C for one hour, followed by a one hour incubation with the detection solution before the fluorescent intensity was measured with an excitation wavelength of 410 nm and an emission wavelength of 480 nm. Data shown are the results from samples assayed in duplicate. These results are provided for demonstration only.

 

Histone Demethylase LSD1 (KDM1) inhibition by Tranylcypromine.
 
Figure 2: Histone Demethylase Assay used to evaluate LSD1 inhibition by Tranylcypromine.

The Histone Demethylase Assay was used to assay activity of the LSD1 positive control in the absence or presence of the irreversible LSD1 inhibitor, Tranylcypromine. 100 µM Tranylcypromine was added to 1 µg LSD1 and pre-incubated for 30 minutes at room temperature. Following the pre-incubation, the enzyme and inhibitor combination was added to the reaction plate containing the recombinant H3K4me2 histone substrate. The enzymatic reaction was incubated at 37°C for one hour, followed by a one hour incubation with the detection solution before the fluorescent intensity was measured with an excitation wavelenght of 410 nm and an emission wavelength of 480 nm. A demethylation standard curve was used to determine the amount of formaldehyde released, which was used to calculate the histone conversion efficiency. Data shown are the results from samples assayed in duplicate. These results are provided for demonstration only.

 

Formaldehyde standard curve for Histone Demethylase Assay Kit.
 
Figure 3: Demethylation standard curve showing fluorescence generated from the formaldehyde standard.

The Histone Demethylase Assay was used to generate a formaldehyde standard curve. The Demethylation Standard was assayed from 0-40 µm formaldehyde. The enzymatic reaction was incubated at 37°C for one hour, followed by a one hour incubation with the detection solution before the fluorescent intensity was measured with an excitation wavelength of 410 nm and an emission wavelength of 480 nm. Results were plotted as a linear regression. Data shown are the results from samples assayed in duplicate. These results are provided for demonstration only.

Histone Demethylase Assay Advantages:

The Histone Demethylase Assay has the advantage of using a Recombinant Histone H3K4me2 protein as a histone substrate instead of a histone peptide. The recombinant protein mimics a native histone substrate, allowing for results that more closely resemble in vivo conditions. As shown in Figure 1, the LSD1 enzyme is able to more efficiently demethylate the included recombinant histone H3K4me2 protein, than a histone H3K4me2 peptide substrate. Therefore, the recombinant histone substrate provided in the Histone Demethylase Assay will allow more accurate analysis of histone demethylation activity.

Comparison of histone H3K4me2 peptide versus recombinant H3K4me2 protein as LSD1 susbstrate.
 
Figure 1: Comparison of histone substrate for LSD1 demethylase efficiency.

The positive control LSD1 enzyme from the Histone Demethylase Assay was used to assay for demethylase activity using either a histone H3K4me2 peptide or the included recombinant histone H3K4me2 protein. One µg of LSD1 was tested with either 70 µM H3K4me2 peptide or with 13 µM recombinant histone H3K4me2 protein. LSD1 was able to convert 73% of the recombinant histone H3K4me2 substrate into formaldehyde, yet it was only able to convert 14% of the H3K4me2 peptide into formaldehdye, even though there was 5-fold more peptide available than recombinant protein for the same amount of LSD1 enzyme.

Why use Histone Demethylase Assay (Fluorescent)?

  • Recombinant histone H3K4me2 protein substrate more closely resembles in vivo conditions
  • Complete assay with optimized buffers for enhanced enzymatic acitivity
  • Includes both positive control LSD1 and a formaldehyde standard
  • Fast procedure can be completed in less than 2.5 hours
  • Easily screen compounds for changes in histone demethylation activity
  • Fluorescent assay can be detected with an excitation wavelength of 410 nm and an emission wavelength of 480 nm

Histone Demethylase Assay (Fluorescent) Method

The Histone Demethylase Assay was designed to detect the formaldehyde released from the reaction of lysine specific demethylase 1 (LSD1, also known as KDM1) with a methylated substrate, H3K4me2. As the LSD1 enzyme demethylates the recombinant H3K4me2 substrate, formaldehyde is released as a by-product. The lysine specific demethylase enzymes are capable of demethylating substrates that are either monomethylated or dimethylated, enabling the production of up to two formaldehyde molecules per substrate. The Detection Reagent reacts with each formaldehyde molecule to generate a fluorescent signal equivalent to the overall production of formaldehyde. The fluorescent signal released upon interaction of the Detection Reagent with the formaldehyde by-product can be measured using a fluorescent microplate reader with an excitation wavelength of 410 nm and an emission wavelength of 480 nm.

The Histone Demethylase Assay Kit can be used to analyze the overall conversion efficiency of an LSD1 sample. Individual conversion efficiencies between the dimethylated histone state and monomethylated histone state cannot be individually determined in this assay. Additionally, the Histone Demethylase Assay Kit can be used to screen compounds for changes in histone demethylation activity. To screen inhibitors, it is recommended to perform a pre-incubation of the LSD1 sample and the inhibitor before proceeding with the rest of the assay protocol.

Flow chart of the fluorescent Histone Demethylase Assay for detecting lysine-specific histone demethylase (LSD1) activity
 
Figure 1: Flow chart of the Histone Demethylase Method.

Sample containing LSD1 enzyme activity is combined with 4X Demethylation Buffer and Recombinant H3K4me2 substrate in a 96-well black half area plate. Enzymatic reactions and detection reactions are carried out at 37°C. Detection Solution is added to each well and the plate is read using an excitation wavelength of 410 nm and emission wavelength of 480 nm. (Click image to enlarge.)

Contents & Storage

The Histone Demethylase Assay (Fluorescent) Kit contains two 96-well black half area plates, plate sealers, Recombinant Histone H3K4me2 protein, 4X Demethylation Buffer, a formaldehyde-based Demethylation Standard, Detection Buffer AM1, Detection Reagent AM1 and a small amount of LSD1 enzyme as a positive control. Storage conditions vary from room temperature to -80°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.