western blotting with anti-goat HRP - bands after ECL but only secondary antibody (Nov/19/2004 )
Hope someone might have some suggestions.....
Done some westerns and found that i am getting bands with just the secondary antibody (anti-goat HRP) after ECL development. Tried different block solutions (Milk, BSA, rabbit serum and FCS) and different secondaries to see if that helps but so far no luck. Main problem is that one of these bands is about the same place as we would expect our band so ideally would like to get rid of these non-specific bands.
Try to treat your secondary Ab with sample, which you load on Western. Of couse, native one, not after boiling in SDS buffer for PAGE.
It could deplet fraction of your secondary Ab reacting with the band.
Some time it works, some time not