| Assay Method Information | |
| | Surface Plasmon Resonance (SPR) |
| Description: | Briefly, a Biacore S200 (Cytiva) at 20° C. and CM5 chips were used for binding analysis. Multi-cycle runs were used for small molecule binding analysis, whereas a single-cycle run was used to measure cyclin binding. The buffer used to prepare the protein samples was 20 mM HEPES, 150 mM NaCl, 10 mM MgCl2, 0.01% Tween 20, pH=7.4). For runs with small molecules, an additional 1% DMSO was added for solubility. GST capture kit conditions (Cytiva catalog number BR100223) were used to capture anti-GST antibody on both the sample and reference cells (7 min immobilization, 10 μL/min flow rate). Both surfaces had high affinity sites capped with an additional 3 min of GST flowed over (5 μg/mL concentration, 5 μL/min flow rate) followed by regeneration (10 mM glycine, pH=2.2). On the subtractive reference surface, GST was immobilized (20 μg/mL, 5 μL/min, 5 min). On the sample surface, GST-tagged CDK2 was immobilized in a similar fashion (50 μg/mL, 5 μL/min, 5 min). |
| Affinity data for this assay | |
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