Multiple unspecifc bands in IP using Protein A sepharose + silver staining - (Mar/14/2006 )

Hi all,

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!.

Hi EthGoh,

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.

Good luck,

Wiz

Hi,

You should pre-incubate your lysate with Protein A beads to reduce the amount inespecific binding to protein A.

tonix37