Western Blot Troubleshooting High Background

Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.

Problem Cause Solution
Blocking issues Too little membrane blocking Increase the concentration of blocking solution
Increase the temperature at which the blocking is performed
Increase the time spent blocking
Blocking proteins reacting with detection system Milk contains biotin; do not use when detecting with avadin-biotin system
Try one of Boster’s other blocking reagents
Contaminated blocking solution Never re-use blocking solutions
Use pure protein as a blocking agent
Incubation issues Excessive substrate incubation Reduce length of substrate incubation
Contaminated incubation equipment Use disposable incubation trays
Thoroughly wash reusable incubation trays between incubations
Insufficient membrane washes between incubations Increase washing stringency
Incubation temperature too high Incubate for longer at a lower temperature
Detection issues Membrane overexposed Check exposure parameters and try a shorter exposure time
Insufficient antibody binding activity Use fresh aliqot of antibody stored at -20C
Avoid thawing and re-freezing antibodies
Store antibodies at -80C for long-term stability
Excessive antibody concentration Reduce antibody concentration
Use a dot-blot test to optimize antibody concentration