Anyone who does western blots routinely knows the feeling: you test a new antibody and are filled with anticipation as your film lurches out of the X-OMAT. Seconds later, your hopes are crushed as you realize your samples have been lost to an ugly blot. Non-specific bands and high background on westerns can be a major source of frustration at the bench. The tips below are designed to help clean up background on your blots.
1. Optimize your antibody concentration
Adding too much antibody can cause blots with high background and non-specific bands. Check the recommended dilution for your primary antibody and consider reducing the concentration of primary antibody. Using excess secondary antibody might also result in blots with high background. Doing a control blot without primary antibody can help determine if your secondary antibody is contributing to non-specific signal.
Optimizing the time and temperature of antibody incubation can also help. Try a shorter incubation time or incubating at 4 degrees overnight instead of room temperature to reduce background.
2. Be sure to block
Blocking is critical for clean blots. Blocking agents such as non-fat dry milk or BSA occupy non-specific binding sites resulting in lower background. Check to see if there is a preferred blocking agent and concentration for your antibody. A 1-5% blocking solution is usually recommended. Block for one hour at room temperature or overnight at 4 degrees with agitation. Blocking solutions should be made fresh as bacterial growth can cause high background. When making the choice between blocking with milk and BSA, remember that milk contains casein, a phosphoprotein that can increase background when used with phospho-specific antibodies.
3. Choose the best membrane for your application and handle with care
The most common membrane choices for western blots are nitrocellulose and PVDF (polyvinylidene difluoride). PVDF membranes are more durable and stand up to stripping and re-probing while nitrocellulose membranes are more brittle and tend to lose signal when stripped and re-probed. PVDF membranes have a higher protein binding capacity than nitrocellulose membranes. This gives them more sensitivity, but also higher background. If your protein is abundant and you do not need to strip and re-probe your membrane, switching to nitrocellulose can help reduce non-specific signal. Auto-fluorescence can also be a source of background when using fluorescent detection systems for western blot. Low-fluorescence membranes can be used to reduce background in this case.
Take care when handling your membrane. Don’t forget to activate PVDF membranes. Make sure that the membrane does not dry out as drying can cause high background. Old membranes can also contribute to background, so be sure to check expiration dates.
4. Wash well
Washing steps in the western blot protocol are essential for high quality blots. Increasing the duration of washing steps as well as the volume of washing buffer can help reduce non-specific staining. Using a detergent, usually Tween-20, is recommended. NP-40 is a stronger detergent that can be used in place of Tween if necessary. If background is still present, a high salt wash can be useful for removing background bands.
5. Take care during detection
Make sure that the detection reagents are mixed well and applied to the blot evenly. Before imaging, remove excess ECL by wicking with a Kimwipe. Use plastic wrap to prevent the membrane from drying out during imaging.