| Assay Method Information | |
| | Gel-based ABPP (LYPLAL1) |
| Description: | For determination of IC50 values for LYPLAL1 inhibitors, the reactions were carried out in 1.5 mL microcentrifuge tubes in a total reaction volume of 25 μL. The final assay mixture contained 50 mM Tris at a pH of 7.5, 0.01% Pluronic F-127, 5% DMSO, and 10 nM of purified LYPLAL1. To 23 μL of LYPLAL1 (10.87 nM) diluted in 54 mM Tris at a pH of 7.5 containing 0.0109% Pluronic F-127 was added 1 μL of compound at 25× the desired final concentration dissolved in 100% DMSO orDMSO (control), and the mixture was incubated at RT for 30 min. To this mixture was added 1 μL of freshly prepared 50 μM FP-rhodamine diluted in 25% DMSO (2 μM FP-Rh, final). The reactions were incubated for 30 min at RT and quenched with 7.5 μL of 4× SDSPAGE loading buffer containing 300 mM DTT, boiled for 2 min at 80°C before 15 μL of sample was loaded per lane, separated by 10% Bis-Tris SDS-PAGE, and visualized in-gel using a flatbed fluorescence scanner (Typhoon Trio). |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |