When processing Western Blots there is substantial evidence that primary antibody and all washing steps should be done at 4 degrees Celsius. Based on our work with both the BlotCycler Touch and Mini we have observed several benefits to running the secondary antibody at 4 degrees during the western blot processing steps.
- Reduced non-specific binding: Lowering the temperature can help to reduce non-specific binding of the secondary antibody to the membrane. This is because the binding of the antibody is slower at lower temperatures, which gives the nonspecific antibodies less time to bind. The only caveat to this is the possible extension of incubation time vs room temperature. The incubation time for the secondary antibody may need to be increased at lower temperatures. This is because the binding of the antibody is slower at lower temperatures.
- Increased signal-to-noise ratio: Running the secondary antibody at 4 degrees can also help to increase the signal-to-noise ratio of the blot. This is because the signal from the specific antibody is more stable at lower temperatures, while the noise from the nonspecific antibodies is reduced.
- Improved resolution: Running the secondary antibody at 4 degrees Celsius can also improve the resolution of the blot. This is because the bands are sharper and more distinct at lower temperatures.
Overall, the benefits of running the secondary antibody at 4 degrees Celsius outweigh the drawbacks in most cases. When working with the BlotCycler, all processing steps are automated so working at 4 degrees has very few if any limitations. Also make sure that you are using a high-quality secondary antibody and wash the membrane thoroughly after incubation.
