| Assay Method Information | |
| | Kinetic Labeling Experiments of Ibrutinib Derivatives with BTK |
| Description: | BTK kinase domain was expressed and purified as previously reported65. Binding experiments were performed in Tris 20 mM pH=8, 50 mM NaCl, and 1 mM DTT. BTK kinase domain was diluted to 2 μM in the buffer, and 2 μM Ibrutinib derivatives were added by adding 1/100th volume from a 200 μM solution. The reaction mixtures, at room temperature for various times, were injected into the LC/MS. For data analysis, the raw spectra were deconvoluted using a 20000:40000 Da window and 1 Da resolution. The signal from masses 20000:30000 and 33000:40000 (which contained no peaks) was averaged and subtracted from the whole signal. The peaks of each species were integrated using a 100 Da window in every direction (reducing the window down to 10 Da did not change the results significantly). |
| Affinity data for this assay | |
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