I'm having an issue in Western blot with an ASC antibody(HY-P80548) as I can't detect any band.
Experimental details: Raw264.7 macrophage cells, concentration around 3 μg/ml, sample volume 8-10 μl; PIPA lysis buffer and PMSF protease inhibitors added.
Dear Marine Ma
which secondary antibody and substrate are you using for the detection?
best
Manuele
Hello,
Are you sure the primary antibody is the problem? Have you made sure the proteins are correctly transfered?
If so, maybe change the anti-ASC antibody? select another antibody? check in the litterature what is the most common protocol to detect this protein?
hi,
- Stimulate cells (LPS/nigericin) to induce ASC expression.
- Switch to stronger lysis (e.g., 1% SDS or urea).
- Load ≥30 µg protein.
- Use overnight antibody incubation + BSA blocking.
- Confirm with a positive control.
- Cross-check with a different ASC antibody if necessary.
Hope this help.
According to PMID: 32560261, RAW264.7 cells do not express ASC, so it’s expected that you wouldn’t detect the target band in your experiment.
The answer to this question comes from MCE Technical Support.
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