| Assay Method Information | |
| | Activation Assay |
| Description: | Using an sGCα 1 gene (NCBI accession No. BC028384.2) and an sGCβ1 gene (NCBI accession No. BC047620.1), an N-terminal FLAG tag-fused sGCα1 and an sGCβ1 expression baculovirus were prepared. These viruses were transfected into insect cells Sf9 (Cat. No. 11496-015, Gibco) to express a protein. From the cell lysates of the insect cells, heterodimers of the N-terminal FLAG tag-fused sGCα1 and sGCβ1 were purified with an M2 Affinity Gel (Sigma-Aldrich, Inc.) to obtain a human sGC.An Example compound was dissolved in DMSO and diluted 20-fold with ultrapure water. 2 uL of the diluted Example compound solution (maximum concentration of 100 uM), 2 uL of a substrate solution [0.5 uM triethanolamine buffer solution, 0.03 uM dithiothreitol, 0.01 uM GTP, 0.04 uM MgCl2, and 0.03 uM sodium nitroprusside (SNP)], and 6 uL of a human enzyme suspension were added to 384-well plates (manufactured by Greiner Bio-One), and incubated at room temperature for one hour. The measurement of the amount of cGMP was carried out, using an HTRF reagent (Cisbio). |
| Affinity data for this assay | |
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