Apoptosis · Antibody review
Caspase-3
Beyotime · AF5132
Target bands and changes are visible despite numerous extra bands; p17 is less prominent at 120 min than at 60 min.
01Experimental images
02Observations
My overall experience
I found this antibody relatively affordable, with visible target bands and a broadly expected trend. However, there are quite a few extra bands, and the cleavage signals are not especially prominent, so the blot takes a little more attention to read.
12-well gel: possible p17 and p19
On the 4–20% gradient gel, apoptotically stimulated lanes 7–12 show a marked reduction in full-length pro-caspase-3. Lanes 10 and 11 have two noticeable bands between approximately 13 and 20 kDa, possibly p17 and p19 based on their positions and changes.
6-well gel: longer is not always better
In the 4–12% gel time course, the approximately 17 kDa band increases at 30 and 60 min, with a clear loss of full-length protein by 60 min. At 120 min, full-length protein is largely gone, but p17 is less prominent than at 60 min. Under these conditions, longer stimulation did not improve the cleavage signal.
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
- 4–12% / 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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