Hello Khemjira Phemphunananchai

These dark spots could arise from impurities. You may have impurities in the blocking solution, or dust or other foreign material on the membrane, or the transfer sponges, paper, and other components can pick up debris and transfer them onto the membrane, resulting in dark spots.

To avoid these dark spots, you should adopt the following measures:

a. Prepare fresh blocking solution and filter it before use. Ensure that the blocking reagent like BSA or milk is dissolved completely or else the clumps may trap the antibody and create dark spots.

b. Ensure that no air bubbles are trapped during the transfer step otherwise it may cause uneven transfer and appear as dark spots on the blot.

c. Sometimes aggregated antibodies can lead to non-specific binding and dark spots on the blot. Aggregates can be removed from the antibody solution by filtering it through a low-retention filter, typically with a pore size of 0.22μm. Avoid repeated freezing and thawing of antibody solution as it can damage the protein and promote aggregation.

d. Dust or dirt on the membrane or in the wash solutions can also lead to dark spots.

e. Ensure your equipment and solutions are clean and dust-free. A dirty scanner or cassette can also contribute to dark spots on the blot. Clean these surfaces regularly.

f. Thorough cleaning of the Transfer pads and transfer boxes is necessary because these can accumulate protein, gel fragments, and components of the transfer buffer over time. This residue can adhere to the membrane, causing dark spots or uneven background. You may soak the transfer pads in 100% methanol for 10 minutes which can help remove accumulated residue. Rinse the blot module with deionized water and then apply 50% nitric acid in deionized water to areas with build-up, which can help clean them.

By proper cleaning of your equipment, using fresh and clean blocking solution and wash buffers, using aggregate-free antibody solution and dust-free membranes, you can ensure a cleaner, more consistent background on your Western Blots, leading to better visualization of your target proteins.

Best,

Hi Khemjira Phemphunananchai ,

do you mean they are visible by naked eye before or after addition of the chemiluminescent solution during blot development? If after, that's simply that there is such strong signal that you can see it with your eye. If you mean before, you can check after each step of your blot preparation, when they appear and that can suggest the problem.

Which brings me to another question - did this appear repeatedly or is it one time thing?

Hi Tomáš Hluska

The yellow spots are visible after adding the chemiluminescent solution, and it just happened this time. The previous blot result is normal.

In that case I'd say it's from the excess of fluorescence. So you need to solve what are those spots. I'd guess wrong blotting (insufficiently dissolved milk?)

block solution is not purified, try BSA or dissolve the skim milk sufficently