Pyroptosis · Antibody review
GSDME NT
ABclonal · A26197
I have had good results with both catalog versions; this test shows clear changes at p35 and approximately 13 kDa.
01Experimental images
Lane assignments
- Lanes 1, 2, 3, 4, 5, 6
- Not recorded
- Lanes 7, 10
- Stimulation (drug not specified)
- Lanes 8, 9, 11, 12
- Stimulation + inhibitor (neither specified)
02Observations
Experience with both catalog versions
I have used both the old and new catalog versions, with good results overall. The older antibody still gave clear bands when I used it again about three years after purchase. The test shown here uses the new version.
p35 and an additional smaller band
Stimulated lanes 7 and 10 show a clear band near 35 kDa, consistent with the expected size of GSDME-NT. There is also a prominent signal near 13 kDa, an interesting feature of this experiment.
Changes after inhibitor treatment
In inhibitor-treated lanes 8, 9, 11 and 12, both the approximately 35 and 13 kDa signals are markedly weaker. I am treating the 13 kDa signal as an additional small band; it changes in the same direction as p35 in this experiment.
03Protein background
GSDME, also called DFNA5, is a gasdermin-family protein with a full-length size usually near 55 kDa. It connects apoptotic signaling with membrane lysis. Caspase-3 cleaves its linker region to release an approximately 30–35 kDa N-terminal fragment, which can form membrane pores and cause pyroptotic lysis in GSDME-expressing cells. Western blots commonly follow loss of the full-length band and accumulation of the N-terminal band. Its apparent migration, intensity and timing vary with cell type, treatment conditions and the region recognized by the antibody.
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
Comments & discussion
No sign-up. Nickname optional.