Pyroptosis · Antibody review
Caspase-1
Proteintech · 22915-1-AP
The early blots were clear. This time, p10 appears with a longer exposure, while other cleavage bands show little difference.
01Experimental images
02Observations
After about three years
I bought this antibody about three years ago. It worked very well at first, with clear pro-caspase-1 and several cleavage bands. The pattern in this experiment differs from those early results.
Short exposure: less precursor and a possible p43 band
At the shorter exposure, the pro-caspase-1 band is weaker, while lanes 7 and 10 show a prominent band between 40 and 55 kDa, possibly p43. Changes in this region are easier to see than the lower-molecular-weight bands.
Longer exposure: p10 becomes visible
With a longer exposure, p10 is visible in lanes 3–6. The other cleavage bands show little difference between groups and are less distinct than in my early experiments.
03Protein background
Caspase-1 is usually present as the approximately 45–46 kDa precursor pro-caspase-1. After inflammasome assembly, the precursor is recruited, dimerizes and undergoes self-cleavage to form active p33/p10. Further cleavage removes the CARD domain and releases p20/p10 from the inflammasome; in cells, this release can reduce its activity. Caspase-1 also processes IL-1β, IL-18 and GSDMD. Western blots may therefore show full-length p45/46 together with fragments such as p33, p20 and p10, depending on the antibody epitope, cell type and sampling time.
04Experimental conditions
- Sample
- Human THP-1 monocytes
- Application
- Western blot (WB)
- Primary antibody dilution
- 1:3000
- Gel
- 4–20% gradient gel
- Electrophoresis
- 200 V, 45 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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