Western Blotting without SDS-PAGE

Lars Hesse larsh at sun0.urz.uni-heidelberg.de
Wed Nov 22 03:56:14 EST 1995


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
Lars




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