SDS-page question - (May/06/2004 )
I've have a purified protein sample. The protein is 25 kDa, but we always see another band at 50 kDa, nevertheless we used reducing conditions, SDS, Mercaptoethanol and we heated the sample. Any suggestions how a kind of a dimer of the protein can still exist after these manipultions?
The protein is demonstrating a hydrophobic aggregate tendency. You might see only 1 band in a 2-D gel. Some reports, vague though, have reported that increasing Ammonium persulfate concentration in the gel pre-mix breaks up these dimers. It all depends on the hydrophobic/glycosylation/modification content of your protein
Do you have some reference about hydrophobic aggregate tendency?
Add b-mercaptoethanol or DTT to all SDS-buffers, or try to reduce disulphids and modify them by iodacetamid e.g., cause it can be disulphid-linked monomers.