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!
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.
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.
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 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.
| Detection Type | Detection Method | Readout | Reference |
|---|---|---|---|
| SAH Detection | Competitive FP Immunoassay | FP | Ref # 1 |
| SAH Detection | Coupled Assay (SAH to AMP) | FP | Ref # 2 |
| SAH Detection | Coupled Assay (SAH to Homocysteine (Hcy)) | FI (Thiol-reactive) | Ref # 3 |
| SAH Detection | Coupled Assay (SAHto H2O2) | FI / Colorimetric (Resorufin) | Ref # 4 |
| SAH Detection | Coupled Assay (SAH to ATP) | Light (Luciferase) | Ref # 5 |
| Methylated Product | Flashplate, Filter-Based Assay (Me-histone, Me-DNA) | Radioactive | Ref # 6, Ref # 7 |
| Methylated Product | Immunoassay; Ab/ELISA (5Me-C) | FI / Colorimetric | N/A |
| Methylated Product | Immunoassay; Ab/ELISA, etc. (Me-Lys) | FI / TR-FRET, etc. | Ref # 8, Ref # 9 |
| Methylated Product | Restriction Enzyme Protection Assay (Me-DNA) | FI / Chemiluminescence | Ref # 10, Ref # 11 |
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.
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* For custom or bulk orders (over 100,000 wells) please contact us (info@bellbrooklabs.com) for a quote.
| Cat. No. | Product Description | Price | Add to Cart |
|---|---|---|---|
| 3023-B | AptaFluor SAH Methyltransferase Assay (100 Assay, 96 Well) | $399.00 | Add to Cart |
| 3023-1K | AptaFluor SAH Methyltransferase Assay (1,000 Assay, 384 Well) | $1,299.00 | Add to Cart |
| 3023-10K | AptaFluor SAH Methyltransferase Assay (10,000 Assay, 384 Well) | $7,399.00 | Add to Cart |
| 3017-1K | Transcreener EPIGEN Methyltransferase Assay (1,000 Assay, 384 Well) | $1,079.00 | Add to Cart |
| 3017-10K | Transcreener EPIGEN Methyltransferase Assay (10,000 Assay, 384 Well) | $6,289.00 | Add to Cart |
| 3017-100K | Transcreener EPIGEN Methyltransferase Assay (10 X 10,000 Assay, 384 Well) | $31,445.00 | Add to Cart |
In addition to our Methyltransferase Assays, Bellbrook Labs also offers the following products and services to aid in the drug discovery process:
BellBrook Labs
1232 Fourier Drive, Suite 115
Madison, Wisconsin 53717 USA
(608) 443-2400
info@bellbrooklabs.com
