I am probing for human LC3B which has two bands with molecular weights of 16 and 14 kDa. However when I image the membrane I see the 16 and 14 size bands but also two more bands around 28 kDa and 24 kDa. What might be possible reasons why I see these higher bands?
The higher molecular weight bands could be dimers. Are you using reducing agents (mercaptoethanol or DTT) during PAGE?
Several possibilities, in addition to Parbhodh's answer:
- If using a polyclonal antibody (e.g. from immunized rabbit antiserum), presence of antibodies that react with unrelated antigens.
- The 28 and 24 kDa species may be truly cross-reactive proteins
- The 28 and 24 kD species may be glycosylated forms of the antigen
My advice would be to try probing the blot with a higher dilution of the primary antibody
Does your protein have any glycosylation site? I do sometimes get higher molecular weight band than the theoretical molecular weight, like Fibronectin N terminal fragment.
The most obvious answer is that they are dimerizing, not necessarily the case but the numbers sure do add up. This is an interesting target, off the top of my head I would think that it could be conjugated with phosphatidylethanolamine(s) and/or other lipid species. It could also be the result of a non-specific binding to polyubiquitin aggregates (ubiquitin ~8.6kDa/ea) due to their close structural homology. I'm not sure what this thing is involved in but I also wouldn't rule out post-translational modification-induced dimerization or aggregation.
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