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| Solution Information | help | |
| Enzyme: | Protein phosphatase methylesterase 1 | |
| inhibitor: | BDBM75707 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this assay is to determine the IC50 values of a powder sample of a test compound for PME-1 inhibition in a complex proteome. In this assay, a fluorophosphonate-conjugated rhodamine (FP-Rh) activity-based probe is used to label PME-1 in the presence of test compound. The reaction products are separated by SDS-PAGE and visualized in-gel using a flatbed fluorescence scanner. The percentage activity remaining is determined by measuring the integrated optical density of the bands. As designed, test compounds that act as PME1 inhibitors will prevent PME1-probe interactions, thereby decreasing the proportion of bound fluorescent probe, giving lower fluorescence intensity in the band in the gel. Protocol Summary: MDA-MB-231 soluble proteome (1 mg/ml in DPBS) was incubated with DMSO or compound for 30 min at 37 C before the addition of FP-Rh at a final concentration of 1 uM in 25 uL total reaction volume. The reaction was incubated for 20 min at 25 C, quenched | |
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