White bands / inverted signal (over-detection) (Western Blot)

Question

Accepted Answer

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. Common causes: 1) Secondary antibody (HRP) concentration too high 2) ECL substrate was overdosed or reaction was too vigorous 3) Exposure was too long; imaging system saturated Solutions: 1) Reduce secondary antibody concentration; increase dilution if needed 2) Reduce ECL substrate amount or switch to a less sensitive substrate 3) Shorten exposure time; use multiple-exposure protocols and pick the best 4) Use non-saturated images for quantification and saving