Western Blotting without SDS-PAGE

M K Archer fr80 at dial.pipex.com
Tue Nov 21 09:03:14 EST 1995


    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.




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