Uneven white spots usually appear on a film, making the western blot background speckled or swirly. The following are Western Blot troubleshooting guide for easy to solve speckled background issues, and can help you get sucessful Western Blotting results.
Speckled background in a Western Blot
|Air bubbles||• During western blot transfer, air bubbles between gel and membrane can cause spots on the film. Use a glass rod as a rolling pin to roll out any bubbles before closing the sandwich cassette.|
|Insufficient washing||• Wash the membrane in a little more washing buffer on an orbital shaker with a little longer washing time.|
|HRP aggregation||• Filter your secondary antibody through 0.2 µm filter membrane to remove any aggregates or just switch to a fresh secondary antibody.|
|Buffer contamination||• Prepare fresh buffers.|
|Mishandling of the membrane||• Minimize the chance of any direct membrane contact with your hands; use clean tools to manipulate it.|