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
上一篇：Western Blot Protocols 下一篇：Western Immunoblotting of Proteins
