White bands / inverted signal (over-detection)

Symptom

White (hollow) bands appear against a black background, or strong positive positions appear white instead of black — signal is so saturated that it appears inverted.

1. 1 Secondary antibody (HRP) concentration too high
2. 2 ECL substrate was overdosed or reaction was too vigorous
3. 3 Exposure was too long; imaging system saturated

1. 1 Reduce secondary antibody concentration; increase dilution if needed
2. 2 Reduce ECL substrate amount or switch to a less sensitive substrate
3. 3 Shorten exposure time; use multiple-exposure protocols and pick the best
4. 4 Use non-saturated images for quantification and saving

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