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Epigenase LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric)

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Suggested Workflow
Nuclear Protein Extraction
 
 
Histone Demethylase Assay
 
Demonstration of high sensitivity of LSD1 activity assay achieved by using recombinant LSD1 with the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Flurometric).
Demonstration of inhibitory effects of an LSD1 inhibitor detected by the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric). [LSD1 concentration: 200 ng/well].
Input Type: Nuclear Extracts, Purified Enzyme
Research Area: Histone Methylation
Target Application: Activity Measurement
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-3079-4848 assays $338.00 
P-3079-9696 assays $549.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

The Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric) is a complete set of optimized reagents, designed for an easy and fast fluorometric measurement of LSD1 activity or inhibition. The antibody-based, immunospecific method directly detects LSD1-converted demethylated products, rather than by- products, in a 96 stripwell microplate format. The kit has the following advantages:

  • 3 hour procedure in a 96 stripwell microplate format allows for either manual or high throughput analysis.
  • Directly measures LSD1 activity via a straightforward detection of LSD1-converted demethylated products, rather than by-products, thus eliminating assay interferences caused by thiol-containing chemicals such as DTT, GSH, and 2-mercaptoethanol.
  • Both cell/tissue extracts and purified LSD1 can be used, which allows for the detection of inhibitory effects of LSD1 inhibitors in vivo and in vitro.
  • Sensitivity is up to 50 times higher than H2O2/formaldehyde release-based LSD1 assays, allowing activity to be fluorometrically detected from as low as 2 ng of purified LSD1 enzyme.
  • Demethylated H3-K4 standard is included, allowing specific activity of LSD1 to be quantified. 
  • Accurate, reliable, and consistent with extremely low background signals.

About Histone Demethylation
Histone demethylation is the removal of methyl groups in modified histone proteins via histone demethylases. The discovery of histone demethylases demonstrates that histone methylation is not a permanent modification, but rather a more dynamic process. One of the most currently studied enzymes of histone demethylation is lysine specific demethylase 1 (LSD1). LSD1 is found to be pivotal in development and differentiation. For example, this enzyme is required to induce skeletal muscle differentiation, and mutation of drosophila LSD1 results in tissue-specific defects in development through disruption of H3-K4 methylation. LSD1 is also shown to participate in regulation of chromatin remodeling, cell death, and global DNA methylation. More importantly, LSD1 is found to be involved in some pathological processes such as cancer and inflammatory diseases. Detection of activity and inhibition of LSD1 would be important in elucidating mechanisms of epigenetic regulation of gene activation and silencing, and benefit cancer diagnostics and therapeutics.

Principle & Procedure
In this assay, a di-methylated histone H3-K4 LSD1 substrate is stably coated onto microplate wells. Active LSD1 binds to the substrate and removes methyl groups from the substrate. The LSD1-demethylated products can be recognized with a specific antibody. The ratio or amount of demethylated products, which is proportional to enzyme activity, can then be fluorometrically measured by reading the fluoresence in a fluorescent microplate spectrophotometer at 530 excitation and 590 emission. The activity of the LSD1 enzyme is proportional to the fluorescent intensity measured.

  Epigenase Kit Other Supplier's Kit
Assay Principle Immunoaffinity-based end product measurement H2O2 or Formaldehyde release-based by-product detection
Assay Format 96-well microplate 96-well microplate
Sensitivity High; 2 ng of enzyme detection limit Very low; 100 ng of enzyme detection limit
S/N Ratio >20 with very low background <4 with high background
Interference by Chemical Reagents None Affected by various detergents, solvents, and irons
Use of Cell Lysates Yes No
Specific Enzyme Activity Quantification Yes No
In Vivo Enzyme Inhibition Assay Yes No
Amount of Substrate Required for Activity/Inhibition Assay

<0.5 µM per assay point

10-50 µM per assay point
Amount of Purified Enzymes Required for Inhibitor Screening 10-50 ng per assay point 200-500 ng per assay point
Accuracy of Enzyme Inhibition Assay High (direct detection of changes in amount of end product) Low (indirect detection of H2O2 or formaldehyde release)

Table 1. Comparison of the Epigenase LSD1 Demethylase Activity/Inhibition Assay Kit and a competing supplier's kit. Competitor data obtained from supplier's website and/or manual.

 
Fig. 1. Schematic procedure for the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric).

Fig. 2. Demonstration of high sensitivity of LSD1 activity assay achieved by using recombinant LSD1 with the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Flurometric).

Fig. 3. Demonstration of inhibitory effects of an LSD1 inhibitor detected by the Epigenase™ LSD1 Demethylase Activity/Inhibition Assay Kit (Fluorometric). [LSD1 concentration: 200 ng/well].

Product Components

LF1 (10X Wash Buffer)
LF2 (LSD1 Assay Buffer)
LF3 (LSD1 Substrate, 50 µg/ml)*
LF4 (LSD1 Assay Standard, 50 µg/ml)*
LF5 (Capture Antibody, 1000 µg/ml)*
LF6 (Detection Antibody, 400 µg/ml)*
LF7 (LSD1 Inhibitor Tranylcypromine, 1 mM)*
LF8 (Fluoro Developer)
LF9 (Fluoro Enhancer)
LF10 (Fluoro Diluter)
8-Well Assay Strips (With Frame)
Adhesive Covering Film
User Guide

* For maximum recovery of the products, centrifuge the original vial after thawing prior to opening the cap.

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Customer Reviews

Rating By j*************@bcm.edu Verified Purchase Reviewed on: Wednesday 14 December, 2016
Application Description
Pros: it is very convenient and consistent.
Cons: It is too sensitive and develop procedure needs to be very careful in case of overexposure.

Procedural Details
SALL4 isoforms and various HA-tagged SALL4 mutants were transfected into HEK293 cells. Cells were then lysed, and anti-LSD1 antibody or IgG control were used for Dynabeads protein G immunoprecipitation (IP). Anti-HA immunoprecipitates were analyzed by Western blotting. C, cells were transfected with different length of SALL4-HA plasmids. Nuclear proteins were extracted and SALL4-LSD1 immunecomplex were pulled down by IgG or anti-HA. LSD1 demethylase activity was fluorometrically quantified with the kit by a SpectraMax M3 microplate reader at 530–590 nm. Error bars represent S.D. of four independent experiments.

Other Thoughts
Reagents (bottles) are easy messed up with some other epigentek kits



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