Apoptosis · Antibody review
Caspase-7
Proteintech · 27155-1-AP
Recent runs have more extra bands, but full-length loss and changes in the smaller bands are still visible.
01Experimental images
02Observations
About three years of use
This antibody gave me good WB results in earlier experiments, but its overall performance was poorer in the two recent runs. I switched from my usual homemade 8% gel to precast gradient gels, omitted blocking, and changed the secondary antibody dilution from 1:8000 to 1:5000; the background increased. These changes may all have contributed. In these runs, gradient gels did not necessarily separate some low-molecular-weight caspase fragments better.
The 12-well gel
On the 4–20% gradient gel, apoptotic stimulation markedly reduced the approximately 35 kDa pro-caspase-7 band; full-length protein was barely detectable in some of lanes 7–12. Two bands appeared near 13 kDa, with one overlapping an additional band. These small bands may reflect further processing or degradation, possibly mixed with nonspecific signals. The approximately 20 kDa band was more prominent in lane 11 with a caspase inhibitor, possibly reflecting an intermediate processing stage. I also saw approximately 20 kDa processing bands in lanes 3 and 4 after LPS + nigericin treatment.
The time course on the 6-well gel
In the time course on the 4–12% gradient gel, the cleaved-fragment band above 13 kDa became stronger at 30 and 60 min, while full-length protein began to fall markedly around 60 min. By 120 min, the full-length band had largely disappeared, but the low-molecular-weight bands showed no further clear increase. With this antibody, I consider the full-length and smaller bands together; sampling time also affects the changes that are visible.
03Protein background
Like caspase-3, caspase-7 is an executioner protease that contributes to apoptotic cell dismantling by cleaving substrates such as PARP. Its precursor is approximately 35 kDa. Processing by upstream caspases removes the prodomain and separates the subunits, producing an approximately 20 kDa large subunit and an approximately 11 kDa small subunit within the active enzyme complex. On western blots, useful features include changes in the precursor and lower-molecular-weight bands over the stimulation time course. The fragments detected vary with 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% / 4–20% gradient gels
- 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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