Reverse Image: White Bands on Dark Background (Western Blot Immunodetection)

Question

Accepted Answer

Film shows negative or reverse image with white or 'bleached' bands on dark background instead of expected dark bands. Also called 'burnt-out' bands. Common causes: 1) Excessive HRP-conjugated secondary antibody concentration 2) Too much HRP activity causing substrate depletion in band regions 3) Localized overexposure in high-signal areas Solutions: 1) Reduce HRP-conjugated secondary antibody concentration 10-fold or more 2) Increase secondary antibody dilution from typical 1:5,000 to 1:50,000 3) Use less sensitive chemiluminescent substrate matched to signal intensity 4) Shorten exposure time to prevent substrate depletion