High Background or Non-specific Bands (Western Blot (Sigma Protocol))

Question

Accepted Answer

Excessive background staining across the membrane or multiple unwanted bands appear Common causes: 1) Insufficient blocking of membrane 2) Primary antibody concentration too high 3) Secondary antibody concentration too high or cross-reacting 4) Inadequate washing steps 5) Antibody incubation times longer than necessary Solutions: 1) Try longer blocking times, higher percentage of blocker, or different blocking agent 2) Check literature for recommended primary antibody dilution and try a range of dilutions 3) Test secondary antibody alone by omitting primary antibody incubation 4) Increase the number or stringency of wash steps 5) Shorten antibody incubation times