Blank smears on western blot - (Jan/20/2006 )

Sometimes my western blots seem to have worked apart fine apart from the appearance of blank (white) smears across the blot. These smears are usually about about 1cm wide and seem to radiate across the blot. In regions of the blot which do not contain the blank smear, normal bands are evident. This problem is really annoying and I don't understand what could be causing it because all washes are done in a volume of at least 30ml.
Please see attached image. Thanks.

can i ask how do u develop your blot?, it maybe related to that

Do you put the ECL directly on the membrane or you put the ECL on a plastic then you cover the drop with your membrane?
The second proposition is cleaner
Maybe it's the problem but i'm not sure at all

strange,

I've heard someone who had some thing maybe similar, it was always on the same place. some in the lab decided not to use the lanes concerned, but he wanted to understand the problem.
in fact it was due to the magnetic stirrer. He decided not to stir anymore during the transfer, and now it is OK.
I was surprised because I was used to stir too, but never had this.


I would say you have also some problems with blocking.
what's your protocol?

I develop the blot with ECL by adding ECL reagent directly onto the membrane, mixing for 1 min and then removing membrane into plastic.

Blocking is performed by incubating the membrane overnight at 4 degrees in 5% milk made up in TBS containing 0.1% tween 20.