Problem with SDS-PAGE-diffuse protein bands - (May/13/2007 )

Dear all,

I have a question about proteins with high mw (185kDa). I run a 10% gel (SDS-PAGE) and after blotting and ab staining I got a signal of my protein of desire, but the bands were diffuse. The preparation of my protein lysate is:
First I wash my cells (adherent) with PBS then I harvest the cells with special extraction buffer from subcellular fractionation kit. (I want to investigate the different cellular localization of my protein). The kit separates the fractions by means of different detergents. Due to the little yield of protein I preciptate the fractions with acetone. After the precipitation I resuspend the pellets with 30µl 2 x SB ( Tris/HCl, Glycerol, 4% SDS, 200mM DTT, bromophenol blue). I always add 5% β-mercaptoethanol to the samples, boiling 95°C 5 min.
What could be reasons for the diffuse bands? Two reducing agents are maybe too much ? And when I did acetone precipitation it seems to me that my proteins pellets vary in their size although I apply 25µg.
Last question: Where is difference in using EtOH or MeOH to equilibrate the pvdf membrane. What is the right treatment? A new colleague uses EtOH, but I have never heard it before.
It would be very helpful for when somebody can answer my question. I am new on this field.
Thanks a lot
Tiffy

I recently had similar diffuse pattern on the same SDS 10% while extracting proteins from HeLa with RIPA buffer.
My mistake was 10 times too much detergent, so I guess it could be this for u.
Detergent was nonidetP40 5% instead of 0.5%