M K Archer wrote:
> 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.
For western and protein-dot-blot analysis we usually use 5% skim Milk (Merck) for blocking the membrane. You can use it in PBS or TBST, dependent on the system (ECL or AP etc.)
Hope it helps