| Assay Method Information | |
| | The TR-FRET Assay |
| Description: | The TR-FRET assay buffer with a formula of 50 mM Tris pH 7.5, 20 mM MgCl2, 0.1 mg/ml BSA and 1 mM DTT was freshly prepared before each experiment. All TR-FRET assays were performed in black 384-well plates with 20 μl/well assay volume in the TR-FRET assay buffer at room temperature (approximately 25° C.). All peptides or compounds were solubilized in DMSO. To test the competitive activity of peptides or compounds against the interaction between GST-MAGEA11 MHD and FAM-PCF11_4 peptide, 10 μl/well peptide or compound at specified concentration was dispensed into a black 384-well low volume plate, followed by 5 μl/well FAM-PCF11_4 (800 nM), 5 μl/well Tb-anti-GST (20 nM) and GST-MAGEA11 MHD (20 nM). The assay components in each well was then mixed by shaking the plate on an IKA MTS 2/4 digital microtiter plate shaker (IKA Works; Wilmington, NC, USA) at 900 RPM for 1 minute. The plate was further centrifuged in an Eppendorf 5810 centrifuge with an A-4-62 swing-bucket rotor (Eppendorf AG, Hamburg, Germany) at 201 g (1000 rpm) for 30 seconds. The plate was then incubated for 90 min with a lid to avoid light exposure to the assay components. After incubation, the fluorescent emission signals at 520 nm and 490 nm channels of individual wells were measured with a PHERAstar FS plate reader (BMG Labtech; Durham, NC, USA) by using a 340 nm excitation filter, 100 μs delay time, and 200 μs integration time. The TR-FRET fluorescence emission ratio (TR-FRET signal) for each well was presented as 10 000×520 nm/490 nm, which was used directly for curve fitting, or further converted to % Inhibition based on the respective positive and negative controls. The graphic software GraphPad Prism 4.81 (GraphPad Software; La Jolla, CA, USA) was used to generate curves and derive IC50 values of tested peptides or compounds, if applicable. |
| Affinity data for this assay | |
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