This report compares the binding of proteins to nitrocellulose membran
es in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7
). Initially, similar amounts of antibodies and other proteins bound t
o the nitrocellulose membrane in both acidic and neutral buffers. Howe
ver, the susceptibility of individual proteins to displacement (stripp
ing) from the membrane by the milk blocking agent depended on the pH o
f the buffer used to bind the proteins to the membrane. Most proteins
that bound to nitrocellulose in acidic buffers were relatively resista
nt to milk-stripping compared to proteins bound in pH 7 buffer. Acid-b
inding of proteins to nitrocellulose also decreased the amount of prot
ein that was stripped from the nitrocellulose membrane when Tween 20 w
as included in the washing buffer. After correcting for the amount of
antibody remaining on the membrane after the milk block, it was found
that acid-bound antibodies were unchanged in biological activity when
compared with the same antibodies bound at neutral pH. These results s
uggest that acid-binding of proteins could increase the sensitivity of
nitrocellulose membrane assays that use milk and/or Tween 20.