Hi Eric, ghost bands (negative staining) is an indication of two much activity on your secondary antibody enzyme (AP, or HRP) which consumes the substrate extremely rapid. Excess of activity of the secondary ab is frequently visible on the membrane with a yellowish faint color appearing at the place of the bands. Try to make a matrix with different combination of amounts of sample versus secondary work from there to find the optimal proportion between sample and ab dilution. The same may have to be done for the primary. Good luck

The proteins may be strongly hydrophobic or bear lipidic moiety. Check pH of transfer buffer also.

Thanks for all your answers so far. But as said before, I do not think those bands are ghost bands. Ghost bands have black borders and bleach in the middle/center, whereas my bands are completely negative/White. I am even more confused as the marker bands show similar patterns where there should be nothing to see at all in chemiluminescence mode. Also my specific bands are visible in regular black quite good, and I don't think that so many other bands are oversaturated that much.

The transfer buffer I use was purchased from the manufacturer of our blotting system, produced specifically for our devices and workflow. Therefire it sould not be a buffer problem. When I use the same membrane with other antibodies after stripping, no such white bands occur, therefore I am thinking of a problem caused by the antibodies.

hi Eric, I was wondering if you have figured out what these white/reverse bands are and/or how to resolve this problem?

A very late update: Just in case you are still wondering, I did not figure the problem out, but I think Artur Burzynski gave a pretty good suggestion above. I changed the antibody and purchased from a different manufacturer, not causing this problem. Thanks for all input!

Finally it was the problem with the Ab.

Could be a problem of too high concentration if second antibody