nonspecific bands on film - (Apr/30/2011 )

Hello everybody,
I am new beginner with western blotting,
I am trying to optimize the western blot for my protein and each case I got many nonsfesific bands after developing the film (even with exposure for 1 min). If I expose more than an hour the nubmer and intensity of bands increase. I am using PBS-0.05% tween and 5% milk as a blotting solution and 1:1000 diluted serum as a primary antibody and 1:3000 2'antibody.
Do you have any suggestion to avoid this?

first, determine if the non-specific bands are from the primary or secondary antibody. then dilute that antibody more or change to a more specific one.

I am sorry for silly questions but how can I determine if it is due to primary or secondary antibody. I mean, is there any way to check this? I am the first petson in our Lab , who is doing western blotting, and I am doing all the procedures by reading on my own, that is why I am very unexperienced:(

you can determine which antibody is responsible by running a duplicate blot without primary. if the non-specific bands show up then the secondary is responsible. if not then the primary is responsible.

after you find out which is responsible, you dilute the responsible antibody until the non-specific bands disappear while the band of interest still shows.

you can also try making the binding conditions more stringent by adjusting salt and/or tween concentrations.