Nonspecific Binding on Western Blot - HELP (Nov/02/2004 )

Hi Everyone,

I am getting a lot of non-specific bands that show up on my western blot .

I am using an anti-his monoclonal antibody that is verified to give almost no background. I am using a goat anti-mouse polycolonal for my secondary Ab.

When I do the HRP detection, I get a lot of non-specific bands that show up on all samples (including the negative control and positive control) and I can't tell if my protein of interest is one of them or not.

I am blocking with 5% dry milk and incubating the antibodies in blocking buffer as well. I am also washing the membrane pretty good. Any suggestions? thanks

Hi,

What is your sample? Is it lysate from eukaryotic cells? Anti-His Ab can has some non-specific binding with eukaryotic proteins. You can try titrating of loading of your sample. And as well play with your anti-His Ab dilution. Try to find lowest amount of your sample for which you still have a signal, very often non-specific band will disappear at this conditions.

Cheers,

Alexei

Yes, I am using lystate from eukaryotic cells. The transfection efficiency in the cell line I am using is usually low, so my protein is probably being expressed at low levels. The western blot shows about non-specific bands in the entire lane and I can't tell if my protein is showing up or not.

Thanks