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EPIgeneous™ Methyltransferase Assay kit

EPIgeneous Cameleon

Introducing a non-radioactive methyltransferase assay for all enzymes and substrates

Imagine being able to use the same biochemical assay for every methyltransferase, reducing assay development time and cost. That’s the simplicity and flexibility you get with the new EPIgeneous™ Methyltransferase Assay from Cisbio. This assay, which directly quantifies S-adenosylhomocysteine (SAH), has been successfully validated on a variety of enzymes and substrates (see table below).

 

Features

  • A universal solution for all methyltransferases and substrates (peptides, nucleosomes, full histones, DNA, RNA, p53 and small molecules)
  • Eliminate false positives with direct detection of SAH (s-adenosylhomocysteine) from SAM (s-adenosylmethinionine) conversion
  • Enzymatic flexibility with a wide range of SAM concentrations 0.4 μM – 200 μM SAM
  • Exceptional sensitivity without radioactivity
  • Easy miniaturization for high throughput screening and profiling
  • Comprehensive, ready-to-use kit

Applications

  • Inhibition analysis
  • Enzymatic reactions in biologically relevant conditions
  • High through put screening and profiling
  • Inhibitor characterization

The EPIgeneous™ Methyltransferase Assay is a universal biochemical assay for all enzymes within the histone (HMTs) and DNA (DNMTs) methyltransferase families that produce S-adenosylhomocysteine (SAH). The methyltransferase activity is assessed by measuring the conversion of SAM (S-5’adenosyl-L-methionine) to SAH. In order to directly measure SAH release, an anti SAH antibody labeled with terbium cryptate and a SAH-d2 tracer are used. The SAH released by the enzymatic reaction competes with the SAH-d2 labeled leading a decrease of the HTRF signal.

The EPIgeneous™ Methyltransferase Assay involves two steps

Enzymatic step

EPIgeneous Enzymatic Detection

The substrate is incubated with the enzyme in presence of compounds. SAM (CH3 donor) is added to start the enzymatic reaction leading to the substrate methylation.

EPIgeneous Enzymatic Detection

Detection step

EPIgeneous Detection

The antibody specific to SAH labeled with Lumi4-Tb competes with both native SAH and d2-coupled SAH. The resulting TR-FRET signal is inversely proportional to the concentration of SAH in the calibrator or in the sample.

EPIgeneous Detection

 

The EPIgeneous Methyltransferase™ assay has been validated with a large set of enzymes and different types of substrates. Enzymatic reactions were carried out at RT or 30°C for 1 to 2h with 0.5 to 10 µM of SAM and related substrates according to the enzymes tested. Reactions were stopped with the addition of detection reagents in 384 low volume plate (20µL) and then read on a BMG labeled Pherastar FS (flashlamp) after 1h incubation.

Methyltransferases Substrates
G9a, EZH2 complex, SET7/9 H3 (1-21) or (1-50) peptide
PRMT1, SET8 H4 (1-25) peptide
DOT1L, SETD2, MLL1 complex Oligonucleosome
SET7/9 p53
DNMT1 DNA (poly(dl-dC))
hN7, WNV, NSP14, NSP10-16 RNA
COMT Dopamine

 

 

EPIgeneous Assay Graph

1. Enzyme titration:

Human recombinant DOT1L was serially diluted in enzymatic buffer from 320nM to 0.001nM and the assay carried out with 10ng/µl (= 77 nM) oligonucleosome as substrate and 2 µM SAM for 2 h at 30°C. The negative controls (no SAM or no nucleosome) show the measurement of the enzymatic specific activity.

A DOT1L concentration of 4.5 nM (EC80) was selected for further experiments. This concentration leads to 10% conversion of SAM into SAH.

EPIgeneous Enzyme graph

 

2. Inhibition effect of SGC0946 on DOT1L assay:

The EPIgeneous™ methyltransferase assay was performed using 0.5 µM SAM (EC40), 77nM oligonucleosome (EC80) and 4.5 nM DOT1L (EC80). The enzymatic reaction was stopped with the detection reagents after 2h incubation at 30°C. IC50 of SGC0946 is in good agreement with the literature (Yu et al. Nature commun., 2012).

As expected, BIX01294 which is a G9a selective inhibitor does not inhibit DOT1L.

Controls of inhibitors without enzyme show that they do not affect the detection reagents.

EPIgeneous Inhibitor graph

 

3. Assay sensitivity:

The graph represents the assay windows obtained with a range of SAM concentrations and at different conversion percentages of SAM into SAH.

The assay was performed using the calibration curve of the kit prepared with the assay diluent

The EPIgeneous™ methyltransferase assay has been optimized to be suitable for a large range of SAM concentrations in the enzymatic step (0.4 – 200µM). With these concentrations of SAM, the assay is able to assess the enzymatic activity with 4 to 10% turnover of the enzyme (hence 4 to 10% conversion of SAM into SAH).

Controls of inhibitors without enzyme show that they do not affect the detection reagents.

EPIgeneous SAM / SAH conversion graph

 

Ordering Info

DescriptionCat. noProduct insertMSDS
Methyltransferase assay 1,000 tests62SAHPEB
Methyltransferase assay 10,000 tests62SAHPEH

Companion products

DescriptionCat. noProduct insertMSDS
SAH calibrator62SAHCLB
-
SAH d2 conjugate - Medium62SAHDLA
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SAH d2 conjugate - Large62SAHDLB
-
Epigeneous detection buffer two - 130 ml62SAHFDD
-
Epigeneous detection buffer one - 24 ml62SAHRDD
-
Ultrapure SAM cofactor (2.4 µmol)62SAHZLC
-
Ultrapure SAM cofactor (22 µmol)62SAHZLD
-




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