western background problem - (Oct/24/2007 )

Hello everyone,

I have a western problem that is bothering me recently. As indicated in the figure, lane 1 is a protein purified under denaturing conditions in 8M urea; lane 2 and 3 are renatured proteins purified with the same protocol(Ni-NTA beads); lane 4 is the total bacterial protein after IPTG induction. The target bands are there. But the background is also very strong in lane 2,3 and 4 (indicated by black arrows). I don't think that this is a problem about milk blocking because lane 1 looks good. In lane 2 and 3, it seems that proteins are stuck at the interface between the stacking gel and the separating gel(as red arrow indicates). So my questions are:how come the background is so strong for some lanes? Does my SDS PAGE gel have a problem? I appreciate your help!

no background in lane 1 because it's a purified protein.
did you try to incubate only with the secondary antibody? It would be better to know which antibody is giving the background.

it is the same protein in all lanes, right? Why I don't see a common band in all the lanes? The 1st lane don't have background also maybe because there is less protein in that sample compared to others. Blocking is thus still a possibility. There are a few other possible reasons, too: antibodies (either 1st or 2nd or both) are not specific; washing.

More protein was probably loaded in lane 4 probably, hence the intense bands. Also there is the possibility that degraded products of the protiens are also being detected by the antibody and your protien might have aggregated leading to higher bands. I donot think the gel has any problem. You could try lower primary antibody conc. to decrease the background.