We are trying to develop a western blot protocol without using
SDS-PAGE and electroblotting. The method involves sucking protein
through a nitrocellulose membrane in a 96-well dot-blot machine (usually
used for DNA/RNA dot-blots). The antibodies are also added into the
wells and sucked through the membrane.
Our problem has been we are unable to block the membrane
sufficiently. The secondary antibody binds to the membrane even if the
primary antibody is not present. We have tried a variety of blocking
agents (BSA, Superblock-Pierce, etc.) but without success. Has anybody
tried this sort of experiment or does anybody have any ideas.