strange bands on western blot - (Jan/10/2006 )

The bands on my western blot appear to be stained periperally at the edge of the band. I loaded 25ug of protein/well and ink stained before probing. Each band is framed by a darker line and the centre of the band is lighter. I have not seen this before.

Any ideas as to what might cause this??

Thanks

MK

my only thought is that it may be like a 'ghost band' or a 'halo band'; I believe this usually happens when there is too much

I would try to load less, and perhaps use a bit less secondary antibody? sometimes the signal is so intense it sort of turns inside-out and I think maybe this is what you are seeing (Pierce's website can explain it better for you)

I agree with aimikins..it most certainily looks like too much of the secondary antibody. You might start by keepinf the protein consatant. I have loaded upto 50 ug and all I needed to do was reduce the secondary antibody concentration.