| Assay Method Information | |
| | Binding of Inhibitors to IAP Protein |
| Description: | The binding affinity of the compounds provided by the present invention to IAP proteins was determined using a fluorescence polarization method (Nikolovska-Colesak et al, Anal. Biochem. 2004, 332:261-73). The recombinant BIR3 domains of human XIAP (residues 238-358), human cIAP1 (residues 255-364) and human cIAP2 (residues 236-342) fusing GST-tags were expressed in E. coli and purified using glutathione agarose 4B affinity chromatography and gel filtration chromatography. A 5-Fam fluorescently modified peptide probe (AbuRPFK(5-Fam)-NH2) was used to test the competitive binding ability of the compounds to the BIR3 domains of XIAP, cIAP1 and cIAP2. The peptide probe (5 nM), XIAP BIR3 (30 nM) or cIAP1 BIR3 (5 nM) or cIAP2 BIR3 (10 nM), and serial diluted compounds were mixed in a test buffer (100 mM potassium phosphate pH 7.5, 100 μg/ml bovine gamma globulin, 0.02% sodium azide, and 1 mM DTT). After incubating the samples at room temperature for 1 hour, the fluorescence polarization values were read using a Tecan microplate reader (FP excitation wavelength 485 nm, absorption wavelength 530 nm). |
| Affinity data for this assay | |
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