Apoptosis · Antibody review
Caspase-9
ABclonal · A18676
Full-length protein and p10 can be detected together; the faint p10 signal is clearer with a longer exposure.
01Experimental images
02Observations
My experience with this antibody
I previously tried caspase-9 antibodies from CST and Beyotime, with fairly average results in my experiments. I bought this one recently, and its main advantage for me is that it detects both full-length caspase-9 and the p10 fragment. There are a few additional bands, but the time-course pattern is fairly clear.
Changes from 60 to 120 min
At 60 min, p10 began to increase clearly while full-length caspase-9 decreased. By 120 min, the full-length band had largely disappeared and p10 increased further. Being able to follow the loss of full-length protein alongside the rise in p10 is one reason I find this antibody useful in this experiment.
A longer exposure for p10
The p10 band is quite faint and becomes clear only with a longer exposure. Although this antibody detects both full-length protein and p10, I still need to pay attention to exposure when reading the blot; a short exposure alone may not reveal the change in p10.
03Protein background
Caspase-9 initiates the mitochondrial apoptosis pathway and has an approximately 46 kDa precursor. Released cytochrome c promotes APAF1 apoptosome assembly, recruiting and activating caspase-9, which then activates caspase-3 and caspase-7. Self-cleavage of human caspase-9 can produce p35/p12; feedback cleavage by caspase-3 can also generate p37/p10 or p35/p10. Western blots may consequently show the full-length protein, large subunits and the p10/p12 small subunits together. Cleavage sites, antibody epitopes and sampling time all influence this pattern.
04Experimental conditions
- Sample
- Human THP-1 monocytes
- Application
- Western blot (WB)
- Primary antibody dilution
- 1:3000
- Gel
- 4–12% gradient 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
- Not recorded
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