Apoptosis · Antibody review
BAK: Two-antibody comparison
Beyotime / ABclonal · AF1591 / A23523
Both antibodies give clear target bands and clean backgrounds; total BAK does not noticeably increase within 2 h.
01Experimental images
02Observations
Good results with both antibodies
I compared Beyotime AF1591 and ABclonal A23523 in this experiment. Both give clear target bands with clean backgrounds and hardly any noticeable extra bands. BAK BYT labels Beyotime, and BAK AB labels ABclonal in the figure.
Total BAK does not rise noticeably
By 2 h, accompanying caspase-3 and GSDME measurements show clear activation and cleavage, but total BAK does not noticeably increase. This contrast is worth noting: the downstream changes are prominent while total BAK remains relatively stable.
03Protein background
BAK is a pro-apoptotic BCL2-family effector located mainly at the mitochondrial outer membrane. Its theoretical mass is approximately 23 kDa, with western blot bands commonly near 23–25 kDa. Death signals can induce conformational changes and oligomerization of BAK, promoting mitochondrial outer-membrane permeabilization, cytochrome c release and the downstream caspase cascade. Because these functional changes involve conformation and assembly, total BAK can remain relatively stable on a conventional denaturing western blot even when apoptotic signaling is already present.
04Experimental conditions
- Sample
- Human THP-1 monocytes
- Application
- Western blot (WB)
- Primary antibody dilution
- 1:3000
- Gel
- 4–12% gel
- Electrophoresis
- 200 V, 35 min
- Transfer
- 300 mA, 30 min
- Membrane
- 0.2 μm nitrocellulose
- Blocking
- 1% PVP, 1 h
- Secondary antibody
- 1:8000, 1 h
- Protein loading
- Not recorded
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