Boniface,

Is this a photo of the actual gel you ran? It looks like different concentrations were loaded in each well. Is that the case?

If your protein is stably forming aggregates in urea, I would try to solubilize it again and then precipitated using ammonium sulfate. I hope it works.

ideally you should not get multimers in SDS-PAGE, it seems your protein polymerization is not di-sulphide based but may be salt bridge based. You can remove salt from the sample and boil it in SDS loading dye for 10 min at 100 deg Celcius prior to loading.

The main cause for multimers is disulfides, so you need to reduce and alkylate to break those bonds and stop them reforming. However, some proteins form multimers that can't be disrupted under any circumstances and this could be one. We have a protein that forms dimers, trimers, all the way to hexamers that can't be disrupted by boiling in SDS or reducing/alkylating. REALLY strong ionic interactions.

As for the lack of signal, you either load more or leave the developing reagent on longer to make more colour. That's the great thing about Western's. Want more signal, leave it longer.

@Maria,

That is the photo of the PVDF membrane. The Idea was to detect the protein and at the same time compare two populations. I loaded 10ug of total protein in each well.

@Andaleeb,

Before loadind the sample I had mixed it with Laemlii buffer and 2-Mercaptoethanol and boiled it for 5 min at 95C.

@Mathew,

The signal is forming. but not at the expected place.

More information

I had done this experiment earlier and got the results at the expected position. The problem was that there was too much streaking and the bands were "fuzzy" and almost formed a line across the membrane. For this experiment I subjected the protein sample through a 2D-Cleanup.