| Assay Method Information | |
| | ICMT Biochemical Assay |
| Description: | An ICMT enzymatic assay was developed using Promega's Methyltransferase-GloTMreagents. In the assay, ICMT catalyzes the methylation of the N-Acetyl-S-farnesyl-L-cysteine by transferring a methyl group from SAM to the farnesylated cysteine and further converts the SAM to SAH. The methyltransferase activity is measured based on the amount of SAH produced from the reaction through the use of coupling enzymes that convert the SAH to ATP. The MTase-Glo detection solution then catalyzes the formation of light from ATP.For the IC50 determination, the compounds were incubated for 30 minutes with 0.04 ug/well ICMT membrane extract from Sf-9 cells. A final concentration of 2.0 μM and 20 μM of SAM and peptide were added and further incubated for 90 minutes at room temperature before adding the MTase Glo and detection reagent. Reaction signals were detected using microplate readers on luminescent mode (Satire Tecan). The IC50 was determined by nonlinear regression, using GraphPad Prism version, 5.03. |
| Affinity data for this assay | |
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