Non specific bands on western blot. - (Jun/20/2008 )

Hi everybody,

I am having some problems with non specific binding with my western blot that has never happened before. So this is what I do:

Load 20ug of protein.

Block in 2% BSA or 5% non fat dry milk for 2 hrs.

Primary Ab(1:1000) incubation is overnight in 5% BSA. Next day wash 3x10'.

Secondary Ab 1:5000 for 1 hr at room temp. wash for 4x10'.

I use ECL as the detection system.

I am getting clear bands for my protein which is Erk (42kda), but I am also getting these other bands below it! Any ideas what is going on, because I haven't had these problems in the past. Your suggestions are very much appreciated the top two bands with the phospho Erk next to it are my bands of interest. [attachment=4870:Erk_1.JPG]

Let's start with a few questions

Haver you changed lot on your abs?
Have you tried bloking only in 5% milk + ab?
At what temperature is your primary antibody incubation?
Have you tried a higher dilution for your secondary antibody (1:10 000) ?

Well, I do have a new lot of Ab, but I am also getting the same problem with my old Ab which hadn't given me any problems before.

The primary Ab dilution is 1:1000, which has worked for me. I haven't gone any higher, because in the past I didn't get any good bands with it. I incubate the primary Ab overnight at 4 C.

Thanks,

Sona.