Detergent is needed for western blot washing solution to help remove nonspecifically bound proteins and antibodies from the membrane. Detergents work by disrupting the hydrophobic interactions between proteins and membranes, which allows them to be washed away. If your wash buffer does not contain a detergent e.g., Tweet this could lead to nonspecific binding and result in high background levels; this can make it difficult to see the specific bands of interest.
The most common detergent used in western blot washing solution is Tween-20. Tween-20 is a non-ionic detergent that is mild and does not damage proteins. Other detergents that can be used in western blot washing solution include Triton X-100 and sodium dodecyl sulfate (SDS). SDS and Triton-X are more harsh and can damage proteins, care should be taken when using these reagents.
The concentration of detergent in the washing solution is important. Too little detergent will not be effective at removing nonspecific binding, while too much detergent can damage proteins. The optimal concentration of detergent will vary depending on the type of membrane and the antibodies being used. A good starting point is to use 0.1% Tween-20 in the washing solution. Depending on the background, the concentration of detergent can be increased to 0.2%.
Some detergents can interfere with chemiluminescence detection; a protocol modification might be needed to adjust for this issue
Here are some guidelines for washing western blots:
- Wash the blots for at least 5 minutes. Using an automated systems will ensure a properly timed protocol.
- Use gentle agitation when washing the blots.
- Change the washing buffer frequently. Precision Biosystems Blotcycler allows for complete automation of all steps: blocking, washing, primary/secondary application and stripping.
- Inspect the blots after each wash to make sure that the background is decreasing.
- Proper washing of western blots will lead to a better signal to noise ratio
