Troubleshooting Guide: Western Blot

Problem: Poor Transfer

Possible Source Test or Action 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 揚oor Transfer?problem for alternatives

Problem: High Protein Binding, Low Signal

Possible Source Test or Action

Problem: Several Bands are Stained

Possible Source Test or Action