Diablo:XIAP Inhibitor Screening Assay Kit
The Diablo:XIAP Inhibitor Screening Assay Kit is an ELISA-based assay designed to measure the binding between Diablo (also named Smac) and E3 ubiquitin-protein ligase XIAP (X-linked inhibitor of apoptosis, also known as mammalian IAP homolog A) for screening and profiling applications. The Diablo:XIAP Inhibitor Screening Assay Kit comes with enough purified Diablo (amino acids 56-239) and XIAP proteins, primary and secondary antibody, assay buffer, and detection reagent for 100 enzyme reactions.
Figure 1. Diablo:XIAP Binding Chemiluminescent Assay Kit schematic.
A 96-well plate is coated with purified Diablo protein. After coating and blocking, XIAP is added in an optimized assay buffer. Next, unbound XIAP is washed away and the plate is incubated with a primary antibody, followed by a secondary HRP-conjugated antibody. Finally, ELISA ECL substrate is added to produce chemiluminescence that can be measured using a chemiluminescence reader. The chemiluminescence signal is proportional to the binding of XIAP to Diablo.
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- 1x PBS (Phosphate Buffer Saline) Buffer
- PBST Buffer (1x PBS, containing 0.05% Tween-20)
- Luminometer or microplate reader capable of reading chemiluminescence
- Adjustable micropipettor and sterile tips
- Rotating or rocker platform
| Catalog # | Name | Amount | Storage |
| 101118 | Diablo, His-Tag* | 2 µg | -80°C |
| 80401 | XIAP, FLAG-Tag* | 1 µg | -80°C |
| Diablo Binding Assay Buffer | 2 x 20 ml | -80°C | |
| Primary Antibody AB31 | 6 µl | -80°C | |
| 52130H | Secondary HRP-Labeled Antibody 1 | 10 µl | -80°C |
| 79743 | Blocking Buffer 3 | 50 ml | -80°C |
| 79670 | ELISA ECL Substrate A (translucent bottle) | 6 ml | Room Temp |
| ELISA ECL Substrate B (brown bottle) | 6 ml | Room Temp | |
| 79837 | 96-well Maxisorp plate | 1 | Room Temp |
* The concentration of protein is lot-specific and will be indicated on the tube containing the protein.
The XIAP (X-linked inhibitor of apoptosis protein), also known as IAP3 or BIRCA4) protein is a RING-containing E3 Ub ligase which can directly regulate caspases and suppress apoptotic cell death pathways. It is considered the most potent member of the IAP family of proteins. It is composed of three BIR (baculovirus IAP Repeat) domains, a UBA and RING domain. It inhibits caspases 3, 7 and 9 by binding to them and targeting them for proteasome degradation via ubiquitination. XIAP can be inhibited by the mitochondrial protein Diablo, also known as Smac (second mitochondrial derived activator of caspases). Diablo is released from the mitochondrial inner membrane space in a caspase-dependent mode, hours after an apoptotic stimulus. An increased expression level of XIAP has been observed in many cancer types and is associated with cancer cell migration by evasion of apoptosis, while mutations in this protein can result in inflammatory bowel disease or X-linked lymphoproliferative disease type 2. The development of Diablo mimetics has been an active area of research, and the use of these molecules alone or in combinatory therapy may prove beneficial in cancer therapy.
Abbas R. and Larisch S., 2020 Cells 9(3): 663.
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