The AptaFluor SAH Methyltransferase Assay utilizes a far-red, TR-FRET end-point readout for direct detection of SAH in a simple mix-and-read format. SAH produced by methyltransferases, such as the NNMT Enzyme (Nicotinamide N-Methyltransferase), drives the assembly of the split aptamer allowing for FRET between a Terbium chelate donor and a DyLight 650 acceptor. The assay provides an excellent tool to screen compound libraries for methyltransferase modulators.
The kit comes complete with the detection reagents required to measure activity. NNMT enzyme is not included in the inhibitor screening assay kit. Please contact us for questions related to acquiring the enzyme.
This assay is designed to be used with purified enzyme preparations. Transcreener assays are not validated with cell lysates, blood, serum, or other biological samples.
Run your enzyme reaction, add AptaFluor reagents, and read your plates. The NNMT assay is compatible with 96, 384, and 1536-well formats.
Z’ measurements using optimized NNMT reaction conditions indicate a robust assay. Robust data like this is vital for sizeable high throughput screens that are difficult to complete due to massive sample quantity. Z’ shown here = 0.73
The assay demonstrates linearity when raw data is converted to SAH using a standard curve. Here, we use both 5 µM SAM and 5 mM Nicotinamide as substrates. The buffer includes 50 mM Tris (pH 8.6) and 1 mM DTT. The reaction occurred for 1 hour at 37ºC prior to adding AptaFluor reagents.
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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 |
|---|---|
| NNMT Enzyme | We have successfully used recombinant human NNMT Enzyme Complex from ProspecBio #ENZ-418. |
| Substrate | Nicotinamide, from Sigma Aldrich #72340. The enzyme reaction contained 5 mM in the 10 μL enzyme reaction. |
| Enzyme Buffer | 50 mM Tris-HCl (pH 8.6) and 1 mM DTT). 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. |
There is extensive crosstalk between the DNA damage response (DDR) pathways and innate immune pathways. Both the individual pathways and the interconnections between them are a focus for exciting new small molecule drug therapeutics that target cancers and debilitating autoimmune disorders. The Transcreener HTS Assay platform accelerates these efforts by providing a robust and easy-to-use biochemical assay to measure activity of key enzymes in the innate immune and DDR pathway.
In this guide, we provide an overview of the DDR and innate immune pathway, and describe Transcreener Assays and Assay Systems for key therapeutic targets. The enzyme targets discussed include:
Nicotinamide N-methyltransferase (NNMT) plays a role in the methylation of nicotinamide, a form of vitamin B3, to N-methyl-nicotinamide. It has gained attention as a potential target for therapy due to its involvement in various biological processes and its possible impact on diseases.
NNMT is involved in the metabolism of nicotinamide, which is a precursor for NAD+ (nicotinamide adenine dinucleotide). NAD+ is crucial for a variety of cellular processes, including energy metabolism and DNA damage repair. Changes in NNMT activity can influence NAD+ levels and thereby affect metabolic functions. For these reasons, NNMT has been implicated in metabolic disorders like obesity and type 2 diabetes. Its involvement in energy metabolism and adipocyte function suggests that altering NNMT activity could impact weight management and insulin sensitivity.
Increased NNMT expression is often observed in several cancers, such as breast cancer, liver cancer, and prostate cancer. It is thought to contribute to tumor progression by altering cellular metabolism and influencing the tumor microenvironment. Targeting NNMT could potentially slow down cancer progression or enhance the effectiveness of other treatments.
NNMT’s effect on inflammation may play a role in neurodegenerative diseases like Parkinson’s Disease and Alzheimer’s Disease. NNMT may also play a role in cardiovascular health by affecting lipid metabolism.
As a promising therapeutic target, NNMT has broad involvement in various disease processes and its impact on metabolism. Developing specific inhibitors could help regulate its activity and potentially reverse or mitigate the effects of diseases in which NNMT is implicated.
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