The PRMT4 Assay determines PRMT4 activity by directly measuring SAH produced during the methylation of SAM. The modified riboswitch that selectively binds with SAH discriminates between SAH and related nucleotides, including SAM (which differs by a methyl group). This produces unambiguous detection of SAH in the presence of excess SAM.
Current non-radioactive SAH detection methods are not sufficiently sensitive to allow detection of many histone methyltransferases (HMTs) using physiological concentrations of SAM. The exquisite affinity and selectivity of the AptaFluor riboswitch overcomes this technical gap, providing screening and profiling of virtually any methyltransferase enzyme with unparalleled sensitivity.
The PRMT4 assay is a simple mix-and-read format. Perform your enzyme reaction, add the detection reagent, and measure. The simplicity of the system yields robust results that also makes it extremely amiable to HTS. It is available with a TR-FRET readout.
A titration of PRMT4 enzyme at initial velocity and SAH formation using 200 nM SAM. Assays were run in LV 384 well white plates, final volume of 20 µL after addition of detection reagents and read on a Pherastar PLUS. PRMT4 enzyme reactions contained 10 µM H3.3 peptide in addition to the indicated amount of PRMT4.
A titration of PRMT4 enzyme at initial velocity and SAH formation using 2 µM SAM. Assays were run in LV 384 well white plates, final volume of 20 µL after addition of detection reagents and read on a Pherastar PLUS. PRMT4 enzyme reactions contained 10 µM H3.3 peptide in addition to the indicated amount of PRMT4.
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| Component | Notes |
|---|---|
| SAM | SAM supplied in the kit should be used for MT enzyme reactions and to create a SAM/SAH standard curve. |
| SAH | SAH is used to create a SAM/SAH standard curve. |
| Enzyme Stop Reagent, 10X | Enzyme Stop Reagent is diluted to 1X in SAH Detection Buffer and used to quench the activity of MT enzyme components. |
| SAH Detection Buffer, 10X | This buffer is used to prepare the SAH Detection Mix and the Enzyme Stop Mix. |
| P1-Biotin | P1-Biotin is a 55-base RNA oligomer with a biotin molecule at the 5' end. It is incubated with TB-Streptavidin to produce P1-Terbium, the donor piece of the SAH aptamer. |
| P2-Dylight 650 | P2-Dylight 650 is an 18-base RNA oligomer with a Dylight 650 molecule at the 3' end. It is the acceptor piece of the SAH aptamer. |
| Tb-Streptavidin | Tb-Streptavidin is used to produce the Tb-labeled (donor) piece of the SAH aptamer. |
| Conjugation Buffer | This buffer is used for the P1-Biotin/Tb-Streptavidin conjugation reaction. |
| Component | Notes |
|---|---|
| PRMT4 Enzyme | We have successfully used recombinant PRMT4 methyltransferase enzyme from Reaction Biology #HMT-11-120. |
| Substrate | We successfully used recombinant Histone H3.3 from Reaction Biology #HMT-11-134. The PRMT4 enzyme reaction contained 10 μM H3.3 peptide. |
| Enzyme Buffer | 20 mM Tris (pH 8.5), 5 mM MgCl2, 1 mM DTT, and 100 mM NaCl. Enzyme buffer components may be optimized as required. |
| Assay Plates | Assay Plates Can Be Purchased Directly Through BellBrook Labs TR-FRET - An entirely white plate with a non-binding surface is required. We suggest Corning 384-well plates Cat. #4513 |
| Plate Reader | A multi-detection microplate reader configured to read TR-FRET is required for the assay. |
| Liquid Handling Devices | Use liquid handling devices that can accurately dispense sub-microliter volumes into 384-well plates. |
| Ultrapure Water | Some deionized water systems are contaminated with nucleases that can degrade both nucleotide substrates and products and RNA aptamers. Careful handling and use of ultrapure water eliminates this potential problem. |
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