Apoptosis · Antibody review
XIAP
ABclonal · A20846
The background is fairly clean; full-length XIAP decreases while an approximately 30 kDa band becomes stronger.
01Experimental images
Lane assignments
- Lanes 1
- 0 min
- Lanes 2
- 5 min
- Lanes 3
- 15 min
- Lanes 4
- 30 min
- Lanes 5
- 60 min
- Lanes 6
- 120 min
02Observations
Band clarity and background
This XIAP antibody worked well overall, with a fairly clean background and only a few additional bands. Changes in both the full-length and lower-molecular-weight bands are easy to follow.
Full-length loss and a growing smaller band
As apoptotic stimulation continues, full-length XIAP gradually decreases while the approximately 30 kDa band becomes stronger. By 2 h, accompanying caspase-3 and GSDME measurements show clear activation and cleavage, and the XIAP time-course pattern is also easy to see.
03Protein background
XIAP is a member of the inhibitor-of-apoptosis protein family. Its theoretical full-length mass is approximately 57 kDa, with western blot bands commonly reported around 53–57 kDa. It directly inhibits caspase-3, caspase-7 and caspase-9 and also has E3 ubiquitin ligase activity, contributing to the regulation of survival and death signaling. During apoptosis, changes in XIAP abundance or processing can appear as loss of the full-length band or the emergence of smaller bands. The fragments and intensities detected depend in part on the region recognized by the antibody.
04Experimental conditions
- Sample
- Human THP-1 monocytes
- Application
- Western blot (WB)
- Primary antibody dilution
- 1:3000
- Gel
- 4–12% gel
- Electrophoresis
- 200 V, 40 min
- Transfer
- 320 mA, 30 min
- Membrane
- 0.2 μm nitrocellulose
- Blocking
- No blocking
- Secondary antibody
- 1:5000, 1 h
- Protein loading
- 15 μL (volume; protein mass not recorded)
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