Functional proteomics strategy for validation of protein kinase inhibitors reveals new targets for a TBB-derived inhibitor of protein kinase CK2

► Unbiased evaluation of CK2 inhibitors using inhibitor affinity Sepharose ► Binding of CK2 to a TBB-related inhibitor is validated in complex mixtures. ► Nucleotide-binding proteins identified as targets for TBB-related CK2 inhibitors. ► Binding to abundant cellular proteins may limit inhibitor bioavailability.