Protocols, optimization tips,
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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.
|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|
Keywords: Western Blot, Troubleshooting, High Background, Nonspecific Staining, Background Noise, Dark BackgroundClick for more troubleshooting tips