Smeared bands for MIDDLE SECTION of the membrane - (Sep/17/2008 )

I blocked the entire membrane in 5% non-fat milk, cut it into 3 pieces (top, middle and bottom) and incubated each in appropriate antibody. After SAME washing conditions and secondary antibody blocking conditions, I exposed the entire membrane at the same time. The results turned out that the top and bottom of the membrane gave good bands (fyi, the bottom one shows good actin bands), but the ONLY the middle section of the blot gave smeared and uncleared bands.

I tried...
- lowering the running voltage from 120V to 100V
- made new primary antibodies
- made new solutions for the gel

Anybody has any ideas about this and what I should do to get good bands?

Thanks a lot.

You're not expecting glycosylation, are you?

If you ponceau stain the middle blot, do the bands look smeared? If so, could have something to do with your transfer.

Do you know if your antibody which you use in the middle section of your membrane gives specific bands? Do you know if your blocking solution is ok for all three antibodies? The problem might not be your gel or transfer but it might be the antibody that you're working with.
Did you stain your gel to see if there is a similar smear in your gel too. This may help to figure out what's going on.

hey - I'd really like to see a pic to aid in diagnosis.

however, I'd be more likely to think it was a problem with transfer.

please show us your blot?

I tried staining the membrane after transfer. It seems there's nothing wrong with the transfer as the bands did not appear 'smeared'. I guess there might be something wrong with the antibody??

how do you handle the membrane when you cut it?

you may be mechanically smearing the blot when you cut it (i've done that, had to figure out a way to cut it without smearing).