Nonspecific Bands Troubleshooting in Western Blots

About western blot antibody non-specific binding problem, multiple bands in membrane as you see, what can one do if there is non-specific binding of primary or secondary antibodies? Reading the following Western Blot troubleshooting guide is supposed to help solving non-specific binding issues in your western blotting.

Western Blot possible cause & solution for nonspecific bands

Nonspecific bands in a western blot

Nonspecific bands in a Western Blot
Possible cause Solution
Non-specific binding of primary antibody • Reduce primary antibody concentration
• Reduce the amount of total protein loaded on gel
• Use monospecific antibody, or affinity (antigen) purified antibodies
Non-specific binding of secondary antibody • Run a secondary antibody only control (w/o primary) and develop blot normally. If background bands appear, use different secondary
• Use monospecific antibody, or affinity (antigen) purified antibodies
Non-specific binding by primary or secondary antibodies • Add 0.1 - 0.5% Tween20 to antibody dilution/wash buffers
• Adjust % of milk in various buffers. Start with 2% non-fat dry milk as Blocking buffer/antibody dilution buffer. Optimize by increasing or decreasing % of milk
• Increase number/time of washes
• Increase NaCl concentration in blocking/antibody dilution/wash buffers to 0.15 - 0.5 M