BellBrook Labs offers two methyltransferase assay kits that monitor the product (S-adenosyl homocysteine or SAH) of methyltransferase reactions. Their universal nature allows both assays to be used with a variety of methyltransferases including protein, DNA, and RNA as well as substrates such as histones, peptides, and nucleosomes.

Explore the AptaFluor SAH Assay and Transcreener Epigen SAH Assay offered by BellBrook Labs!

Methyltransferases as Therapeutic Targets

Methyltransferases are a diverse family of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM or AdoMet), the second most common enzymatic cofactor after ATP, to amino, thiol, or hydroxyl groups of acceptor molecules. This generates S-adenosylhomocysteine (SAH) as a byproduct. 

Methyltransferases play a role in epigenetic regulation through methylation of histones at lysine and arginine residues and methylation of DNA at cytosines in hemi-methylated CpG sites. In humans, there are over 50 Protein Lysine Methyltransferases (PKMTs), at least 10 Protein Arginine Methyltransferases (PRMTs), and three DNA Methyltransferases (DNMTs).

Together these enzymes play a critical role in the dynamic modification of chromatin, and they are increasingly being targeted for cancer and other diseases with an epigenetic component.

Methyltransferase Assays at BellBrook Labs

AptaFluor SAH Methyltransferase Assay

The AptaFluor SAH Methyltransferase Assay uses a naturally occurring aptamer, or riboswitch, that selectively binds with SAH, the invariant product of methyltransferase reactions. The exquisite affinity and selectivity of the riboswitch combined with a positive TR-FRET signal enable screening and profiling of histone methyltransferases with unparalleled sensitivity.

Split aptamer directly detects SAH resulting in a positive TR-FRET signal

Transcreener EPIGEN SAH Methyltransferase Assay

The Transcreener EPIGEN SAH Methyltransferase Assay provides universal methyltransferase detection in an HTS-proven format. It combines the extensively validated Transcreener AMP²/GMP² Assay with coupling enzymes that convert the SAH produced in a Methyltransferase reaction to AMP for detection with a fluorescent polarization (FP) readout.

The coupling enzyme converts SAH to AMP. Transcreener AMP2/GMP2 Assay detects this with an FP readout

Comparing BellBrook's Methyltransferase Assay Kits

1Continuous interference of 1.3% with AptaFluor and 0.8% with EPIGEN assays detected in sample libraries tested.

2Continuous mode can only be used when using a peptide as a substrate.

How Does a Biochemical Methyltransferase Assay Work?

The Methyltransferase Assay methods rely on either detection of the methylated product or detection of SAH. There are several formats and readout options for both detection methods (Figure 1). 

Detection of SAH

Assay methods that detect SAH formation have the advantage of providing universal detection of MT enzymes regardless of the acceptor substrate or the mix of methylated reaction products.  Universal assay methods reduce drug candidate assay development costs by using one single set of assay reagents for all methyltransferase targets.

Direct Detection

The new AptaFluor SAH Methyltransferase assay allows for direct detection of SAH using an aptamer. By strategically splitting aptamer, excellent assay sensitivity can be achieved. It also allows for direct detection with a positive TR-FRET readout. Direct immunodetection of SAH would also be advantageous as it would eliminate the potential for compound interference from coupling enzymes, however, it requires an antibody that specifically binds SAH in the presence of excess SAM; ie, that differentiates on the basis of a single methyl group.  There is one literature report of an FP-based methyltransferase assay using an anti-SAH antibody from a diagnostic assay kit for homocysteine, but its commercial availability is unknown.

Coupled Assays

Coupled enzyme assays have also been developed for SAH detection. For instance, SAH can be converted to homocysteine and adenosine using SAH hydrolase, and homocysteine is then detected using covalent reaction with a thiol-sensitive fluor. Alternatively, the adenine portion of SAH can be converted to urate by the sequential action of three coupling enzymes, with co-production of hydrogen peroxide in the final step. Hydrogen peroxide can be detected colorimetrically or fluorescently, via formation of resorufin. In addition, a luciferase-based coupled assay relies on the sequential conversion of SAH to adenine, AMP, and ATP, with the final step catalyzed by pyruvate phosphate dikinase. BellBrook Labs’ EPIGEN SAH Assay utilizes two coupling enzymes to convert the SAH to AMP, which can then be measured with a monoclonal antibody and tracer with negligible cross-reactivity with SAM.  This assay format can detect both histone methyltransferases and DNA methyltransferases.

Methyltransferase Assays Table

Detection TypeDetection MethodReadoutReference
SAH DetectionCompetitive FP ImmunoassayFPRef # 1
SAH DetectionCoupled Assay
(SAH to AMP)
FPRef # 2
SAH DetectionCoupled Assay
(SAH to Homocysteine (Hcy))
Ref # 3
SAH DetectionCoupled Assay
(SAHto H2O2)
FI / Colorimetric
Ref # 4
SAH DetectionCoupled Assay
(SAH to ATP)
Ref # 5
Methylated ProductFlashplate, Filter-Based Assay
(Me-histone, Me-DNA)
RadioactiveRef # 6, Ref # 7
Methylated ProductImmunoassay; Ab/ELISA
FI / ColorimetricN/A
Methylated ProductImmunoassay; Ab/ELISA, etc.
FI / TR-FRET, etc.Ref # 8, Ref # 9
Methylated ProductRestriction Enzyme Protection Assay
FI / Chemiluminescence
Ref # 10, Ref # 11

Detection of Methylated Products

The most quantitative and reliable assay method are filter-based or flash plate-based radioassays that use 3H-SAM to generate 3H-methylated products.  However, the associated regulatory and disposal costs are a liability for High Throughput Screening (HTS).  Immunoassays for methylated lysine, arginine and cytosine have been used for both PKMT and DNMT enzyme assays, either in an ELISA format or in a homogenous format such as TR-FRET.  Antibody selectivity is critical for this approach and can limit the utility of the assay, as some histone methyltransferases can generate both mono- and di-methylated lysine products, and the known antibodies do not recognize both forms.  Assays based on enzymatic cleavage (or protection) of products have also been applied to both PKMTs and DNMTs utilizing methylation state–dependent restriction enzymes or endoproteinase Lys C, which is unable to cleave at methylated lysine residues.  Although application of this approach has generally relied on a solid phase method such as ELISA, or a separation step, a more HTS-friendly fluorescence de-quenching configuration was recently developed for DNMT1.


Jack of All Trades and Master of Everything?, W.A.M. Loenen. (Journal Abstract), Department of Toxicogenetics, Division 5, Leiden University Medical Centre, Building 2, T-03-011, Einthovenweg, Leiden, The Netherlands. Biochem Soc Trans. 2006 Apr;34(Pt 2):330-03.(PubMed Abstract).

Epigenetics, an Emerging Target Class for Drug Screening. Comley J, Drug Discovery World, Spring, 2011.

Chromatin Modifications and Their Function. Kouzarides T, Cell 2007; 128:693-705.

DNA Methyltransferases as Targets for Cancer Therapy. Ghoshal K, Bai S, Drugs Today (Barc) 2007; 43:395-422.

Protein Methyltransferases as a Target Class for Drug Discovery. Copeland RA, Solomon ME, Richon VM, Nat Rev Drug Discov 2009; 8:724-732.

Biological Rationale for the Use of DNA Methyltransferase Inhibitors as New Strategy for Modulation of Tumor Response to Chemotherapy and Radiation. Gravina GL, Festuccia C, Marampon F, Popov VM, Pestell RG, Zani BM, Tombolini V, Mol Cancer 2010 Nov 25; 9:305.

Unsafe SETs: Histone Lysine Methyltransferases and Cancer. Schneider R, Bannister AJ, Kouzarides T. Wellcome/Cancer Research UK Institute and Department of Pathology, Tennis Court Road, Cambridge, UK, CB2 1QR. Trends Biochem Sci. 2002 Aug;27(8):396-402. (PubMed Abstract)

Epigenetics and Airways Disease. Adcock IM, Ford P, Ito K, Barnes PJ. Airways Disease Section, National Heart and Lung Institute, Imperial College London, UK. Resp Res. 2006 Feb 6;7:21 (Full text)

Protein Methyltransferases as a Target Class for Drug Discovery. Copeland RA, Solomon ME, Richon VM, Nat Rev Drug Discov 2009; 8:724-732.

Epigenetics in Cancer. Sharma S, Kelly TK, Jones PA, Carcinogenesis 2010 Jan; 31(1):27-36.

Molecular Recognition of Sub-Micromolar Inhibitors by the Epinephrine-Synthesizing Enzyme Phenylethanolamine N-Methyltransferase. McMillan FM, Archbold J, McLeish MJ, Caine JM, Criscione KR, Grunewald GL, Martin JL, J Med Chem 2004; 47:37-44.

Structural Basis for Inhibition of Histamine N-Methyltransferases by Diverse Drugs. Horton JR, Sawada K, Nishibori M, Cheng X, J Mol Biol 2005: 353:334-344.

RNA Modifications: a Mechanism that Modulates Gene Expression. Karijolich J, Kantartzis A, Yu YT, Methods Mol Biol 2010; 629: 1-19.

A Universal Competitive Fluorescence Polarization Activity Assay for S-Adenosylmethionine Utilizing Methyltransferases. Tiffany L. Graves, Yi Zhang, and John E. Scott. Department of Pharmaceutical Sciences, Biomanufacturing Research Institute and Technology Enterprise, North Carolina Central University, Durham NC 27707, USA. Anal Biochem. 2008 Feb 15; 373(2):296-306 (Abstract)

Development and Validation of a Generic Fluorescent Methyltransferase Activity Assay Based on the Transcreener AMP/GMP Assay. Klink, TA, Staeben M, Twesten, K, Kopp AL, Kumar M, Schall Dunn R, Pinchard C, Kleman KM, Klumpp, Lowery RG, JBC 2011, submitted.

A Coupled Fluorescent Assay for Histone Methyltransferases. Collazo E, Couture JF, Bulfer S, Trievel RC., Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, 48109-0606, USA. Anal Biochem. 2005 Jul 1;342(1):86-92. (Abstract).

An Enzyme-Coupled Continuous Spectrophotometric Assay for S-Adenosylmethionine-Dependent Methyltransferases. Dorgan KM, Wooderchak WL, Wynn DP, Karschner EL, Alfaro JF, Cui Y, Zhou ZS, Hevel JM. Department of Chemistry, Washington State University, Pullman, WA 99164, USA. Anal Biochem. 2006 Mar 15;350(2):249-55. (Abstract)

An Enzyme-Coupled Ultrasensitive Luminescence Assay for Protein Methyltransferases. Ibáñez G, McBean JL, Astudillo YM, Luo M. Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA. Anal Biochem. 2010 June 15;401(2):203-10. (Abstract)

A Continuous Protein Methyltransferase (G9a) Assay for Enzyme Activity Measurement and Inhibitor Screening. Dhayalan A, Dimitrova E, Rathert P, Jeltsch A, J Biomol Screen 2009; 14:1129-1133.

Continuous Enzymatic Assay for Histone Lysine Methyltransferases. Rathert P, Cheng X, Jeltsch A, Biotechniques 2007; 43:602, 604, 606 passim.

Small Molecule Regulators of Protein Arginine Methyltransferases. Cheng D, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT, J Biol Chem  2004; 279:23892-23899.

Reversal of H3K9me2 by a Small-Molecule Inhibitor for the G9a Histone Methyltransferase. Kubicek S, O'Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA, Kelly TA, Jenuwein T, Mol Cell 2007; 25:473-481.

Accessing Protein Methyltransfease and Demethylase Enzymology Using Microfluidic Capillary Electrophoresis. Wigle TJ, Provencher LM, Norris JL, Jin J, Brown PJ, Frye SV, Janzen WP, Chem Biol 2010 July 20; 17:695-704.

Fluorescence-based High-Throughput Assay for Human DNA (Cytosine-5)-Methyltransferase 1. Ye Y, Stivers JT, Anal Biochem 2010 June 1; 401:168-172.

A Fast and Efficient Method for Quantitative Measurement of S-Adenosyl-L-Methionine-Dependent Methyltransferase Activity with Protein Substrates. Brenda B. Suh-Lailam and Joan M. Hevel. (Journal Abstract). Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322, USA and Center for Integrated Biosystems, Utah State University, Logan UT 84322, USA. Anal Biochem. 2010 Mar 15; 398(2):218-24.

Kinetic Analysis of Human Protein Arginine N-Methyltransferase 2: Formation of Monomethyl- and Asymmetric Dimethyl-Arginine Residues on Histone H4. Ted M. Lakowski and Adam Frankel., Division of Biomolecular and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, The University of British Columbia,  Vancouver, BC, Canada, V6T 1Z3. Biochem. J. 2009 June 26; 421(2):253-61 (Full Text).

A Nonradioactive DNA Methyltransferase Assay Adaptable to High-Throughput Screening. Youn-Hi Woo, P.T. Ravi Rajagopalan, and Stephen J. Benkovic. 414 Wartik Laboratory, Department of Chemistry, Pennsylvania State University, University Park, PA 16802, USA. Anal Biochem. 2005 Mar 13; 340:336-340. (Abstract).

An Enzyme-Coupled Colorimetric Assay for S-Adenosylmethionine-Dependent Methyltransferases. Hendricks CL, Ross JR, Pichersky E, Noel JP, Zhou ZS. Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213, USA. Anal Biochem 2004 Mar 1;326(1):100-05. (Abstract).

A Real-Time Assay for CpG-Specific Cytosine-C5 Methyltransferase Activity. Wood RJ, McKelvie JC, Maynard-Smith MD, Roach PL. School of Chemistry, University of Southampton, Southampton, Hampshire, SO17 1BJ, UK. Nuc Acids Res. 2010 May; 38(9):e107. (Full Text).

Epigenetics: Tools and Technologies. Janzen WP, Wigle TJ, Jin J, Frye SV, Drug Discov Today Technol. 2010 Spring; 7(1):e59-e65; 7:e59-65.

Small Molecule Regulators of Protein Arginine Methyltransferases. Cheng D, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT, J Biol Chem 2004; 279:23892-23899.

Reversal of H3K9me2 By a Small-Molecule Inhibitor for the G9a Histone Methyltransferase. Kubicek S, O'Sullivan RJ, August EM, Hickey ER, Zhang Q, Teodoro ML, Rea S, Mechtler K, Kowalski JA, Homon CA, Kelly TA, Jenuwein T, Mol Cell 2007; 25:473-481.

State of the Arg: Protein Methylation of Arginine Comes of Age. Anne E. McBride 1 and Pamela A. Silver; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115USA. Cell 2001 July; 106:5-8. (Full Text).

Gene-Specific Targeting of H3K9 Methylation is Sufficient for Initiating Repression In Vivo. Andrew W Snowden, Phillip D Gregory, Casey C Casea and Carl O Paboa. Sangamo Biosciences, Point Richmond Tech Center, 501 Canal Boulevard, Suite A100, Richmond, CA 94804 USA. Curr Biol. 2022 Dec; 12(24):2159-66. (PubMed Abstract).

Substrate Specificity and Kinetic Mechanism of Mammalian G9a Histone H3 Methyltransferase. Patnaik D, Chin HG, Esteve PO, Benner J, Jacobsen SE, Pradhan S, J Biol Chem 2004; 279:53248-53258

Functional Analysis of Genetic Variation in Catechol-O-Methyltransferase (COMT): Effects on mRNA, Protein, and Enzyme Activity in Postmortem Human Bain. Chen J, Lipska BK, Halim N, Ma QD, Matsumoto M, Melhem S, Kolachana BS, Hyde TM, Herman MM, Apud J, Egan MF, Kleinman JE, Weinberger Dr. Clinical Brain Disorders Branch, Genes, Cognition, and Psychosis Program, National Institute of Mental Health, National Institutes of Health, Bethesda, MD 20892, USA. AM J Hum Genet 2004 Nov:75(5):807-821. (PubMed Full Text).

Purification and Characterization of Steptomyces Griseus Catechol-O-Methyltransferase. Dhar K, Rosazza JP. Division of Medicinal and Natural Products Chemistry, Center for Biocatalysis and Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242, USA. Appl Environ Microbiol 2000 Nov;66(11):4877-4882. (PubMed Full Text).

Multiple Molecular Forms of Catechol-O-Methyltransferase. Evidence for Two Distinct Forms, and Their Purification and Physical Characterization. Huh MM, Friedhoff AJ. The Millhauser Laboratories, New York University School of Medicine, New York, New York 10016. J Biol Chem. 1979 Jan 25;254(2):299-308.

Catechol-O-Methyltransferase (COMT)-Mediated Methylation Metabolism of Endogenous Bioactive Catechols and Modulation by Endobiotics and Xenobiotics: Importance in Pathophysiology and Pathogenesis. Zhu BT, Curr Drug Metab 2002; 3:321-349.

InVitro Characterization of the Enzyme Properties of the Phospholipid N-Methyltransferase PmtA from Agrobacterium Tumefaciens. Meriyem Aktas and Franz Narberhaus. Microbial Biology, Ruhr-University, Bochum, Germany: Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und Biotechnologie, Ruhr-Universität Bochum, Universitätsstrasse 150, NDEF 06/783, D-44780 Bochum, Germany. J Bacteriol 2009 April:191(7):2033-2041. (PubMed Full Text).

BellBrook’s SAH Methyltransferase Assay Kits

* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.

In addition to our Methyltransferase Assays, Bellbrook Labs also offers the following products and services to aid in the drug discovery process: