| Solution Information | help | |
| Enzyme: | Serine/threonine-protein phosphatase PP1-alpha catalytic subunit | |
| inhibitor: | BDBM61233 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this counterscreen assay is to determine whether compounds identified as active in a set of previous experiments entitled, "Fluorescence-based primary biochemical high throughput screening assay to identify inhibitors of Protein Phosphatase 5 (PP5)" (AID 1987) are nonselective due to inhibition of PP1. This assay determines the PP1 inhibition potency of tested compounds. This biochemical, fluorescence-based assay employs purified PP1alpha (catalytic subunit) and 3-O-methylfluorescein phosphate (OMFP) as the substrate. Cleavage of OMFP by PP1alpha converts OMFP to the fluorescent product 3-O-methylfluorescein. As designed, compounds that act as PP1alpha inhibitors will block PP1alpha catalytic activity, thereby decreasing OMFP cleavage and production of fluorescent product, resulting in reduced well fluorescence. Compounds are tested in triplicate in a 10-point 1:3 dilution series starting at a nominal test concentration of 67 micromolar. Protocol Summa | |
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