| Assay Method Information | |
| | Testing the Affinity of DB Compounds to PSMA Protein Using Biacore |
| Description: | Table 5: The affinity of DB compounds to PSMA protein was tested using the Biacore 8K (Cytiva) instrument to detect ligand binding to PSMA protein (Sino Biological). PSMA protein was captured on an SA chip. Before fixing the ligand ( flow paths 1 and 2, flow rate of 10 μL/min), PSMA protein (10 μg/mL, flow rate of 5 μL/min, injection time of 600 s) was fixed on flow path 2 using a flow buffer. The sensor surface was adjusted by continuously injecting 1 M NaCl three times in 50 mM NaOH. After each ligand injection, additional cleaning was performed using isopropanol in 1 M NaCl and 50 mM NaOH (flow paths 1 and 2, flow rate of 10 μL/min, injection time of 60 s).[0170]All compounds were dissolved in 100% DMSO and diluted to 10 mM, then further diluted to the appropriate highest concentration in the analysis buffer (PBS, PH 7.4, 1 mM TCEP (tris-(2-hydroxyethyl) phosphine), 0.05% P20, 2% DMSO). The analysis was conducted under the following conditions: 15° C. analysis temperature; analysis steps=all set to LMW kinetics; cycle type=single cycle (90 s contact time, 1800 s dissociation time, 30 μL/min flow rate, flow paths 1 and 2); detection in flow=path 2-1. Data evaluation was performed using Biacore Insight Evaluation Software, fitting the data to a 1:1 binding model. |
| Affinity data for this assay | |
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