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Western Blot troubleshooting

Zoom+  Zoom- Publish date: 2014-06-27   Hits: 1748
Western blotting troubleshooting guide
Western blot troubleshooting tips - protein transfer problem: poor transfer efficiency, air bubbles trapped when assembling the transfer sandwitch (use a tube or roller to get ride of any trapped air bubbles), voltage and current is not optimized for the size of protein of your interests. - film development problem: try service and clean the film developer. try film that is optimzed to the signal strength produced by the band of your membrane. - non-specific bands on western blot: try to use a more specific primary antibody and use optimzed concentration of antibody (in some cases, an used antibody saved from previous run works fine). Blocking is not sufficient and should try overnight blocking at 4c in 5% dry milk (non fat) or BSA solution. Make sure protein lysate is prepared properly without proteolytic degradation (keep lysate on ice or storage in -80c, always use protease inhibitors cocktail). - High background problem: film exposed too long, too high concentration of secondary antibody, insufficient wash of membrane or strength of detergent in washing buffer. - Low signal problem: low abundant protein of your interests, primary antibody is not working, secondary antibody too low, washing too stringent. In some cases, a gel documentation system is helpful to capture weak bands. You can also try more sensitive chemiluminescent system which are commercially available from several good vendors. This troubleshooting guide discuss following problems: (Agrisera) High background signal Strange/Non-specific bands on the blot Low sensitivity Is a right band detected? Uneven results with lots of spots Winning Westerns: 12 Proven Strategies to Optimize Your Western Blots
 
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