Pyroptosis · Antibody review
GSDMD NT
ABclonal · A24059
The p30 NT band is prominent, and its reduction after inhibitor treatment is easy to see.
01Experimental images
Lane assignments
- Lanes 1, 2, 7, 8, 9, 10, 11, 12
- Not recorded
- Lanes 3, 4
- Prominent p30 NT bands; treatment not recorded
- Lanes 5, 6
- Inhibitor added (not specified)
02Observations
A clear p30 NT signal
This antibody worked very well in this experiment. Lanes 3 and 4 show a prominent GSDMD p30 NT band at approximately 30–31 kDa, making the target signal easy to pick out.
A clear reduction with inhibitors
After inhibitor treatment, the p30 NT bands in lanes 5 and 6 are markedly weaker. Comparing these lanes makes the treatment-related difference clear, which I was particularly pleased with.
03Protein background
GSDMD is an executor of pyroptosis, with a full-length protein usually around 53–55 kDa. Cleavage by inflammatory caspases releases an approximately 30–31 kDa N-terminal fragment, which oligomerizes and forms membrane pores that allow cellular contents to escape. Caspase-3/7 can cleave GSDMD at a different site, generating an approximately 43 kDa inactive fragment. Thus, p30/31 and p43 reflect different processing routes. Antibodies recognizing the full-length protein, its N-terminal region or a specific cleavage site can produce 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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