Multiple unspecifc bands in IP using Protein A sepharose + silver staining - (Mar/14/2006 )
Hope you can help.
I been getting really high unspecific background binding with my sepharose protein A beads during IP of nuclear fractions. Multiple bands are seen with beads only control. I incubate ab for 2 hours with beads followed by 3 hours with lysate. Been trying to optimise it for some time now, but nothing seems to work.
Any suggestions will be greatly appreciated!.
You should definitively try to pre-incubate your lysate with Protein A beads for about 1 hr. This should reduce the amount of contaminants binding to your beads. Blocking your beads with BSA (0.5% in TBS or PBS) could also help you.
You should pre-incubate your lysate with Protein A beads to reduce the amount inespecific binding to protein A.