Universal MTase-Glo™ scheme and sensitivity of SAH detection.

(A) Schematic diagram of the basic principle of MTase GloTM. Basically, any MTase (MTase) that is capable of methylating its substrate and S-Adenosylmethionine (SAM) will generate methylated substrate and S-Adenosylhomocysteine (SAH), the latter is a universal product for all MTases. Conversion of SAH to ADP and to ATP is performed by coupled enzyme reactions. The ATP produced can be quantitated by a luciferase/luciferin bioluminescent reaction. (B) A standard curve showing the relative luminescence units (RLU) in response to SAH concentrations, inset show a lower range of SAH concentration vs. RLUs. Reactions were assembled with the indicated concentrations of SAH in a low-volume 384-well white solid plate. The MTase-Glo™ Assay was performed as described in Materials and Methods. Data were collected using a plate reading luminometer (INFITIE M1000PRO, TECAN). Each point represents average of four data points; the error bars represent the standard deviation. Data analysis was performed with Excel, Microsoft Office 365. (C) Table representing the data generated to quantify SAH concentrations and calculating signal to background and signal to noise ratio from panel B.