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

Question

Accepted Answer

Film shows 'bleached' or 'burnt-out' white bands against dark background instead of expected dark bands. Bands appear as negative images where high protein concentration exists. Common causes: 1) Excessive HRP-conjugated secondary antibody concentration causing substrate depletion 2) Very high target protein concentration combined with high antibody levels 3) Prolonged chemiluminescent substrate incubation with high HRP levels Solutions: 1) Reduce HRP-conjugated secondary antibody concentration (e.g., increase dilution 10-fold from 1:5000 to 1:50000) 2) Decrease protein loading amount on gel to reduce target antigen concentration 3) Shorten substrate incubation time or reduce primary antibody concentration