Strange Western blot background - (Oct/28/2010 )

Hello!

I have encountered a strange Western blot background issue recently.

I find that there is always a high background on top of the membrane, which disturbs me to observe my target band.
I don't think it is the issue of blocking or washing, as the background is always localized on the top.
Besides, I play around with the ECL development step, like applying the reagent on top/bottom/side of the membrane first, changing the volume of reagent added, remove all excess reagent thoroughly, but I still get the same result.
Moreover, it is not the problem of the film or the developing machine because I have exposed my membrane 3 times on the same film on different area, and only the top of the membrane shows high background.

My primary antibody is homemade antiserum raised from rabbit and we do not purify it, so it is expected to give some non-specific bands.

Here is my prodecure:
1) Gel: 10% separating (with stacking), 75V before stacking, 100V after stacking
2) Transfer: semi-dry, 15V, 1.5 hr with PVDF membrane
3) Block with 5% milk in TBST (0.05% tween-20), 1 hr at room temperature
4) 1:1500 1st Ab in blocking, 4C overnight
5) Wash with TBST 4 times, 10 min each
6) 1:10000 2nd Ab (Goat Anti-rabbit IgG-HRP) in TBST, 1 hr at room temperature
7) Wash with TBST 6 times, 10 min each
8 ) Develop with ECL plus (GE healthcare), expose for 5s, 15s, 30s

Do anyone encounter the same problem before? How can I solve it? My labmate suggests me to run my target band to the center to avoid the high background region, but I still want to know what the problems are. Thank you.


Exposure time: 30s
The arrow indicates my target band.
The two center lanes (with white bands) are protein ladder

You could try diluting your primary and/or secondary even more. Or try blocking in BSA instead of milk. Some ab work better this way.

Also, 30s is pretty short time for exposure, which means you get a very strong signal. Try either to load less protein or higher dilution for your Abs.

Good luck!

Thanks.

I will try to optimize the dilution. I find that if it is the problem of high concentration of Ab, the background is usually everywhere, but not just the top, so I suspect that there are some other reasons behind this.