Troubleshooting Guide: Western Blot

Use alternate buffer system (such as CAPS buffer pH 10.5) with a higher pH.

Assemble transfer stack with additional membrane soaked in cathode buffer on the cathode side of gel. This will capture proteins that migrate towards the cathode. Identify membrane location in stack and immunostain all membranes in stack Adding 0.005 - 0.01% SDS to cathode buffer can increase transfer efficiency of protein from gel.

Note: Increased SDS concentration may interfere with binding to membrane.

Problem: High Background Staining Possible Source Test or Action Optimize the blocking step by trying alternate blocking solutions (e.g., non-fat milk, gelatin, etc.) or increasing time and temperature of blocking step.

Problem: Low Protein Binding Possible Source Test or Action Keep the length of TTBS washes to a minimum See or Transfer?problem for alternatives.

Problem: High Protein Binding, Low Signal Possible Source Test or Action.

Problem: Several Bands are Stained.