That smearing and a band that does not enter the gel is a normal result of cross-linking conjugation which we usually observe when we conjugate our protein-antigen with a carrier protein for immunization. We never see any individual bands for dimer, trimer, tetramers and so on. The smearing represents random interactions of different amino acids on the surface of the protein resulting in dimer, trimer, tetramer and so on, each of them have multiple bands on gel due to the random interaction and as a result different mobilities of each particular dimer isomer (as well as trimer, tetramer and so on). The band that does not enter the gel represent all oligomer isomers with the highest MW.

It sounds like the crosslinking experiment was successful in that crosslinking occurred, such that all the monomer was consumed. The question is, what products resulted? If the crosslinker concentration was relatively high, extensive non-specific crosslinking could result in mixed aggregates, some even large enough that they won't run into the gel. Is there a relatively low range of crosslinker concentrations at which low-order oligomers (e.g. dimers, trimers, tertamers) are seen? If so, this might give you reason to expect that such oligomers exist in the absence of crosslinker.

Another way to study oligomerization without crosslinking is through analytical gel filtration (size exclusion) chromatography, preferably with a multiangle light scattering detector. Dynamic light scattering and analytical ultracentrifugation can also be used if the equipment is available.

Thank you for the insight. SEC-MALS and DLS are probably where this is headed, but I just wondered what the common explanations for the disappearing (diffusing) protein were. I tested a series of crosslinker concentrations until I was within the dynamic range for the known dimer, as judged by the shift in MW to the expected size. Simultaneously, I tested my mystery protein but all I see is smearing and a band that doesn't enter the gel. This is unexpected.

That smearing and a band that does not enter the gel is a normal result of cross-linking conjugation which we usually observe when we conjugate our protein-antigen with a carrier protein for immunization. We never see any individual bands for dimer, trimer, tetramers and so on. The smearing represents random interactions of different amino acids on the surface of the protein resulting in dimer, trimer, tetramer and so on, each of them have multiple bands on gel due to the random interaction and as a result different mobilities of each particular dimer isomer (as well as trimer, tetramer and so on). The band that does not enter the gel represent all oligomer isomers with the highest MW.

Thank you Viktor. So I either have a high molecular weight oligomer or aggregated protein. Now I will run analytical gel filtration and look for evidence of aggregation, followed up by DLS.