Hello Dongwoo Choi

There could be two possibilities in this case:

1. In the experimental group, if the target protein is cleaved post-translationally and if one uses polyclonal antibody, then multiple bands will be observed because of the ability of polyclonal Ab to recognize multiple epitopes.

2. Alternate splicing of mRNA can result in multiple protein products. If the epitope which the antibody recognizes is shared between the proteins, then too multiple bands can be observed.

Best Wishes.

Another possible explanation is that the antiserum you are using (assuming it is polyclonal) is not sufficiently specific and is detecting other proteins besides the one you are interested in. If you have a sample of an extract from the same source that does not have any of the antigen, then you can pre-adsorb the antiserum with it to remove the antibodies that recognize the extra bands.

Are those proteins from whole extracts or after e.g. immunoprecipitation? If they are after IP, you can see antibody chains in some cases.

The other issue: yes, if you use too high concentration of the secondary antibody, you can obtain non-specific detection - the problem analogous to what Adam B Shapiro explained above.