High Background Signal on Western Blot (Western Blot Immunodetection)

Question

Accepted Answer

Overall high background across entire membrane reduces signal-to-noise ratio. Specific bands difficult to distinguish from background noise. Common causes: 1) Secondary antibody concentration too high causing nonspecific binding 2) Secondary antibody cross-reactivity with endogenous proteins in sample 3) Insufficient washing volumes or times after antibody incubations 4) Protein-protein interactions on membrane surface 5) Film overexposure with chemiluminescent detection 6) Immobilon-PSQ membrane requires higher blocking reagent concentration due to greater surface area Solutions: 1) Increase secondary antibody dilution (typically 1:5,000 to 1:20,000) 2) Use secondary antibody pre-adsorbed with serum from target species 3) Increase wash volumes and times; use SNAP i.d. vacuum system to pull wash solution through membrane 4) Add Tween-20 (0.05%) to wash and detection buffers to minimize protein-protein interactions 5) Shorten film exposure time; use less sensitive detection reagent matched to signal intensity 6) For Immobilon-PSQ membrane, add 0.5M NaCl and 0.2% SDS to wash buffer and extend wash to 2 hours