bands fuse together - (Nov/26/2011 )

Hi,

I've done a western blot on HO-1 protein and got a normal well separated bands using HO-1 antibody or Actin antibody but the bands became fusing together for subsequent trial run using the same types of buffers and reagents. Protein amount: 30ug for each lane. Since the deep blue ladder made the membrane dark, I've run the ladder out of the gel to the buffer. Does it affect the bands?
many thanks.

Bands fusing meaning sample in Well 1 is too close to sample in Well 2?