| Assay Method Information | |
| | SPR Binding Assay |
| Description: | IRAK4 protein. N-terminal His-TEV-AVI tagged catalytical domain of human IRAK4 (a.a. 163-460) was co-expressed with Bir A in insect cells, and purified to >95% homogeneity by a combination of Ni-NTA affinity chromatography, ion-exchange and size-exclusion chromatography. Phosphorylation and mono-biotinylation of purified IRAK4 were confirmed by mass spectrometric analysis.IRAK4 SPR. IRAK4 SPR was set up on Biacore T200 or S200 by using Biotin CAPture kit (Cytiva). In brief, purified IRAK4 in capture buffer (25 mM Hepes, 150 mM NaCl, 1 mM TCEP, pH7.4) was captured onto a CAP sensor surface via the interaction of biotin to streptavidin Typical capture level is between 1,000 RU to 2,000 RU, Compound binding kinetics to IRAK4 was examined with running buffer (25 mM Hepes, 150 mM NaCl, 1 mM TCEP, 2% DMSO, pH7.4). Serially diluted compounds were injected at 50 μl/min in single-cycle for 60-s association of each injection followed by 360-s dissociation at the end. |
| Affinity data for this assay | |
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