Apoptosis · Antibody review
Cleaved caspase-3 p12
ABclonal · A28349
p12 appears at the expected position, increases clearly after stimulation and decreases or disappears with inhibitors.
01Experimental images
Lane assignments
- Lanes 1, 2, 3, 6, 9, 11, 12
- Not recorded
- Lanes 4
- LPS + nigericin
- Lanes 5
- LPS + nigericin + inhibitor (not specified)
- Lanes 7, 10
- Apoptotic stimulation (drug not specified)
- Lanes 8
- Inhibitor added; exact treatment combination not recorded
02Observations
A good p12 result
I was pleased with this p12 antibody. The band is at the expected position and increases clearly after apoptotic stimulation in lanes 7 and 10. In inhibitor-treated lane 8, the signal is markedly reduced or absent.
Some p12 with LPS + Nigericin as well
Alongside the apoptotic conditions, lane 4 treated with LPS + Nigericin shows a small amount of p12. The signal disappears in the corresponding inhibitor-treated lane 5, an interesting difference when comparing the two lanes.
03Protein background
Caspase-3 is an executioner protease in apoptosis and is usually present as an approximately 32 kDa precursor. Processing promoted by upstream caspase-8 or caspase-9 generates the p12 small subunit and the p19 large subunit, which can mature further into p17. Activated caspase-3 cleaves substrates including PARP and GSDME. Western blots commonly track the full-length protein and p19/p17 or p12. Because these fragments come from different regions of the protein, antibodies directed against the large subunit and those directed against p12 often show different band patterns.
04Experimental conditions
- Sample
- Human THP-1 monocytes
- Application
- Western blot (WB)
- Primary antibody dilution
- 1:3000
- Gel
- 4–20% 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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