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Assay Method Information

Assay Name:  Gel-based ABPP (CES1)
Description:  For determination of IC50 values for CES1 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, 5% DMSO, and 1.75 μg of CES1 lysate transiently expressed in COS7. To 23 μL of CESμ1 (1.75 μg of lysate) diluted in 54 mM Tris at a pH of 7.5 was added 1 μL of compound at 25x the desired final concentration dissolved in 100% DMSO or DMSO (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 4x SDS-PAGE loading buffer containing 300 mM DTT and boiled for 2 min at 80 °C before 15 μLof 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
 

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Last update November 1, 2007
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