Hi

It could be that your 1ry abs are detecting variants of yr ptn or immature forms (e.g. pre-pro or proprotein) suggesting that the ptn is synthesized and stored in the ventricle in its immature form prior to release but no atrium.

Regards,

Bassem

There are a few possibilities: 1) Different protein modifications, 2) different isoforms, and 3) if bands are smaller than expected, proteolytic cleavage of your protein. If you think it is phosphorylation, you can CIP-treat your lysate before running it on a gel. In case of a negative results, there are quite a few other modifications that can shift a protein on a gel (ie ubiquitin, roughly induce a 8 kDa shift). For different isoforms, you could perform a good old Northern blot. Alternatively, you could mass spec the different band.

In the atria and septum (the tissues that give you a doublet rather than a triplet), are all three phosphorylation events known to occur? Perhaps the phosphorylation state is tissue-dependent. 

I'd probably start with phosphatase treatment, to make sure that all the forms you are seeing collapse down to a signal band. If thorough phosphatase treatment does NOT give you a single band, there's probably another post-translational modification involved (Ub, glycosylation, etc).

It's possible that you DO have all three phosphorylation states in the atria and septum lysates, but some other factor is affecting gel mobility. Another factor might be causing two of the phospho-forms to migrate very similarly on the gel, so that the two forms generate only one band.