| Assay Method Information | |
| | BIR Binding Assay |
| Description: | The BIR domain binding assays are FRET based competition assays that utilize His-tagged versions of each of the BIR2 and BIR3 domains from cIAP1, cIAP2, and XIAP (each domain assayed separately) each at a custom optimal concentration and a probe, 200 nM SMAC/DIABLO peptide AVPIAQKSE labelled with AlexaFluor647 (part #crb 1110326h, Discovery Peptides). The assay is conducted in 50 mM HEPES, 150 nM NaCl, 1 mM CHAPS, 5% Glycerol, 1 mM DTT, in dI water with a final pH of 7.4 and final volume of 20 uL. Final protein domain concentrations are: 50 nM cIAP1-BIR3 (Part #APT-11-370 Reaction Biology), 100 nM XIAP-BIR3 (Part #APT-11-351 Reaction Biology), 200 nM XIAP-BIR2 (Part #APT-11-470 Reaction Biology), 325 nM cIAP2-BIR2 (Part #APT-11-489 Reaction Biology), 50 nM cIAP2-BIR3 (Part #APT-11-372 Reaction Biology), 325 nM cIAP1-BIR2 (Part #APT-11-487 Reaction Biology). Compounds were plated using a HP Tecan D300 printer in a final total volume of 202 nl and DMSO concentration normalized across the 15-point, 3-fold dilution series. Peptide and probe were prepared at 2× relative to the concentrations above in binding buffer, 10 uL added to the prepared compound plates, and incubated for 60 minutes. LANCE Eu-W1024 (part #AD0401 Perkin Elmer #, vendor) was prepared at 2× concentration in binding buffer for final concentration of 2 nM, 10 uL added to each well, and plates incubated for 30 minutes. Plates were read on an ENVISION multifunction plate reader with 320 nM laser excitation to obtain the 615 nm/665 nm emission ratio as indicative of probe:protein proximity. |
| Affinity data for this assay | |
|---|---|
| If you find an error in this entry please send us an E-mail | |