Excessive background signal across the membrane obscures specific bands, caused by non-specific antibody binding, inadequate blocking, or improper handling.
Common Causes
1Secondary antibody non-specific binding or cross-reaction with sample species; primary antibody concentration too high
2Secondary antibody binding to blocking reagent (e.g., phospho-specific antibodies reacting with casein in milk)
3Insufficient blocking (3–5% non-fat milk, BSA, or normal serum for 1 hr at room temperature recommended)
4Inadequate washing between steps leaving residual unbound antibodies; Ponceau S autofluorescence in fluorescent detection
5Excessive substrate concentration or incubation time with enzyme-conjugated antibodies; substrate sensitivity too high
6Membrane dried out during incubation, contaminated during handling, or multiple membranes incubated facing each other
Solutions
1Run control without primary antibody; use secondary antibody from different species than sample; try pre-adsorbed secondary antibody
2For phospho-specific antibodies, block with BSA instead of milk; add mild detergent (Tween 20) to incubation and wash buffers
3Increase blocking incubation to 1 hr at room temperature with 3–5% non-fat milk, BSA, or normal serum; consider changing blocking agent
4Wash extensively with buffer between all steps; remove Ponceau S before fluorescent immunostaining
5Dilute substrate and reduce incubation time; replace with milder substrate; reduce signal amplification (e.g., less biotin conjugation)
6Keep membrane covered in buffer to prevent drying; maintain clean membrane; place multiple membranes back-to-back; reduce exposure time