Please give us some more details on antibodies used, species they are from, band size you have on the gel. Is there a control lane without sample to precipitate?

Wolfgang Schechinger hi, I’m using rabbit antibody against SMN. The first lane is Input, second lane is IgG rabbit. Third and fourth is the pull-down proteins. I am getting background every time. I even increased the number of cells and it’s the same.

Hi Sonia Marina

What is the protein loading conc. and loading volume?

I suppose the loading conc. is very high. Are these tissue or cell samples?

Thanks,

Samir Ranjan Panda loading volume 25 ul. They are from cell samples

Which species is your secondary made in?

Seems to be a cross reaction between the secondary and the rabbit IgG. You could try to covalently immobilize the antibody for the IP on the beads using a bifunctional crosslinker or resort to a biotinylated primary and streptavidin beads.

Wolfgang Schechinger my secondary Ab is rabbit

You mean, it's anti-rabbit, right? Which species is it made in?

After incubation with primary antibody overnight try soaking the membranes in TBST for 30 mins followed by washing while rocking for one hour OR vice versa. this will decrease the back ground Sonia Marina