Pyroptosis · Antibody review
GSDMD
CST · Cell Signaling Technology · #39754
A favorite after about three years of use, with clear p30 and p43 processing patterns.
01Experimental images
Lane assignments
- Lanes 1, 2
- Not recorded
- Lanes 3, 4
- LPS + nigericin
- Lanes 5, 6
- LPS + nigericin + inhibitor (not specified)
- Lanes 7, 10
- Apoptotic stimulation (drug not specified)
- Lanes 8, 9, 11, 12
- Apoptotic stimulation + inhibitor (neither specified)
02Observations
An antibody I keep using
I bought this GSDMD antibody about three years ago and have continued using it. What I particularly like is being able to detect processing associated with both pyroptosis and apoptosis using the same antibody.
p30 under pyroptotic conditions
After LPS + Nigericin treatment, lanes 3 and 4 show very clear GSDMD p30 NT bands. In inhibitor-treated lanes 5 and 6, the p30 signal is markedly weaker.
p43 under apoptotic conditions
In apoptotically stimulated lanes 7 and 10, full-length GSDMD is barely visible and p43 is clear. In inhibitor-treated lanes 8, 9, 11 and 12, p43 decreases and full-length signal increases, giving an easy-to-read contrast.
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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