Based on the description and the provided image, it appears that there is intense non-specific staining at the bottom of the nitrocellulose blot. This staining is likely due to background signal caused by non-specific binding of the secondary antibody. Here are a few suggestions to help reduce background staining:

1. Optimization of blocking: Increase the blocking duration or concentration to minimize non-specific binding. You mentioned using ThermoFisher Superblock, so you may try increasing the concentration or incubation time to enhance blocking efficiency.

2. Wash steps: Ensure that you are performing sufficient and thorough wash steps between antibody incubations. Increase the number of washes and the duration of each wash to reduce background signal.

3. Primary antibody concentration: Optimize the concentration of the primary antibody. Use a range of dilutions to determine the optimal concentration that provides specific staining without increasing non-specific background.

4. Secondary antibody selection: Consider using a different secondary antibody that is highly specific for the primary antibody species and has minimal cross-reactivity with other proteins. This can help reduce non-specific binding.

5. Incubation conditions: Evaluate the temperature and duration of the incubation steps. Adjusting the temperature and time can sometimes help reduce non-specific binding.

6. Blocking reagents: If the current blocking reagent is not effectively reducing background staining, try alternative blocking reagents such as BSA (bovine serum albumin) or milk.

Regarding the suggestion to cut off the bottom of the blot before staining, it can be an option if the intense staining is limited to that region and does not interfere with the analysis of the specific bands. However, it is generally recommended to address the underlying cause of background staining rather than removing portions of the blot.

By optimizing the blocking, washing, antibody concentrations, and incubation conditions, you can often reduce background staining and improve the specificity of your immunoblotting results. It may require some trial and error to find the optimal conditions for your specific experiment.

All the best

The bright spots are something in your samples and not a blotting artifact. My best guess is that it is just an accumulation of small peptide fragments from proteolysis of the proteins in your lysate. They give a strong signal because they may include peptides recognized by your primary (or secondary) antibody.

You might try adding protease inhibitors very early in the preparation of your samples and see if that helps.

Michael J. Benedik thank you for your suggestions. After washing plates twice with ice cold PBS, I introduce M-PER (lysis buffer) containing both protease and phosphatase inhibitors at the recommended concentration. I hope the proteins are not fragmenting in this case.

I neglected to mention that these bands don't show up if I use secondary HRP followed by enhanced chemiluminescence, just the secondary alexa dye-conjugated antibody.

I switched secondary antibodies and the bright band disappeared! It seems that the dye at the dye front has something that is fluorescent in the Alexa 647 filter set. The same samples and membrane, when stained with Alexa 488, does not show the intense fluorescence.

I will continue to use Alexa 647 as an alternative dye only if the proteins are large enough to be quite distant from the dye front.