Table 2. Troubleshooting guide for Western blot issues.

Problem	Potential causes	Solutions
Weak or no signal	(1) Inefficient transfer to nitrocellulose (2) Low antibody binding (3) Excessive washing	(1) Extend transfer time to 1.5 h, or increase methanol content to 25% in transfer buffer (2) Optimize primary antibody dilution (e.g., 1:2000) and incubate for 48 h at 4°C (3) Reduce TBST wash time to 3−4 min per wash
High background noise	(1) Incomplete blocking (2) Cross-contamination during washing	(1) Block with 5% BSA instead of milk for phospho-specific antibodies (2) Increase TBST washing frequency (e.g., 3 × 10 min) and use fresh buffers
Uneven band intensity	(1) Uneven gel loading (2) Poor membrane contact	(1) Load equal protein amounts (e.g., 20–25 μL per lane) (2) Ensure even pressure in transfer sandwich