| Assay Method Information | |
| | SPR Binding Assay |
| Description: | Table A2: In vivo biotinylated PRMT5-MEP50 was diluted to 4.5 μM in 25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, and 0.05% Tween-20 and injected at 5 μl/min flow rate into flow cell 2 (FC2) of a Series S Sensor Chip SA (Cytiva) in a Biacore T200 or in a Biacore 8K plus (Cytiva). SPR screening was performed in MTA running buffer (25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, 20 μM MTA, 0.05% Tween-20 and 2% DMSO). The biotinylated PRMT5-MEP50 surface was equilibrated with MTA running buffer for 12 hours prior to the start. The test compound affinity was determined using multi-cycle injection of each fragment from 0.001 to 500 μM over the PRMT5•MTA at a flow rate of 30 μl/min and with association and dissociation times of 20 and 60 seconds respectively. PRMT5•MTA surface activity was confirmed at the initiation, and the end of the run by titration of EPZ015666 (KD=11 and 13 μM respectively). Subsequently, compound titration was repeated in SAM-running buffer (25 mM Bicine pH 7.6, 100 mM NaCl, 1 mM TCEP, 20 μM SAM, 0.05% Tween-20, and 2% DMSO). The PRMT5•SAM surface was equilibrated for at least 5 hours prior to compound titration and the PRMT5•SAM surface activity was confirmed at the end of the fragment titration run by titration of EPZ015666 (KD<1 nM). After double referencing, the steady-state response was extracted for each fragment concentration and was fit to the Langmuir isotherm equation to determine the equilibrium dissociation constant (KD). |
| Affinity data for this assay | |
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