Help, odd pattern - pic included - (May/06/2010 )

Hello everyone -- I am completely stumped and desperate for advice on some Western troubleshooting that has been occuring for nearly a month now.

Heres the story:

I am performing western blots using mainly bio-rad mini equipment.
I was getting excellent results for the longest time, but recently my tubulin, and sometimes, other antibodies around the same MW as tubulin are showing uneven intensities - even when I load the same exact sample (same amount) in every lane in the gel.
Everything used to work great and I haven't changed anything and all of a sudden this occured.

Some other info.
I am doing a tank transfer, 10% MeOH @ 100V for 1 Hr w/ ice block on stir plate.
Blocking with Bio-rads Non-fat milk at 5% in TBST, Antibodies also diluted in Blocking.
Primary overnight in cold room on shaker 1:40,000, Secondary 1:40,000 for 1 hour.
Have tried both PVDF and Nitrocellose - same problem.
Have borrowed another labs transfer equipment - same problem
Have prepared all fresh buffers - same problem
Another key fact - other antibodies on these same blots are coming through with consistent intesity so it appears the problem is only occuring with tubulin or around that MW on the membrane.

Let me know if I can provide other details.

TONS of appreciation in advance for any ideas.

It appears to be a transfer or antibody issue... transfer issues, perhaps not enough pressure in the tank, or some damaged pads. Antibody - perhaps the membrane is not getting completely covered by solutions, this could be due to a bump in the dish you are using for incubations or something similar.

If it is happening only around that size, then it is something you are doing of using consistently - check how you are placing things in dishes, blot modules, etc. It definitely does not look like a loading issue.

i had the same problem before! also tried everything u described - new buffer, new abs, borrowed equipment from other labs etc. finally it was resolved when i changed to new set of sponge during transfer step.

hope this helps. good luck!

resolved it - weirdest thing, I lied actually, the one thing I did change when this started to happen was stirring the transfer on a stir plate... before I just kept the transfer box cold and on ice,, well I went back to that in desperation and that was the issue... not sure if the transfer was getting to hot or if something with the stirring was actually disturbing the transfer ( i was stirring very slow - 50rpm) but thats what happened, and the disturbances were near the lower half of the gel... close to the stir plate, but I guess near the middle of the transfer so maybe the heat was more intense there. Anyway.. crazy stuff.