title: Antibodies bound to nitrocellulose in acidic buffers retain biological activity.
abstract: This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in both acidic and neutral buffers. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on the pH of the buffer used to bind the proteins to the membrane. Most proteins that bound to nitrocellulose in acidic buffers were relatively resistant to milk-stripping compared to proteins bound in pH 7 buffer. Acid-binding of proteins to nitrocellulose also decreased the amount of protein that was stripped from the nitrocellulose membrane when Tween 20 was 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 suggest that acid-binding of proteins could increase the sensitivity of nitrocellulose membrane assays that use milk and/or Tween 20.
authors.affiliation: Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas.
authors.forename: W L
authors.initials: WL
authors.lastname: Hoffman
authors.forename: A A
authors.initials: AA
authors.lastname: Jump
authors.forename: A O
authors.initials: AO
authors.lastname: Ruggles
authors.forename: P J
authors.initials: PJ
authors.lastname: Kelly
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D002021:Buffers; D003101:Collodion / Q000378:metabolism*; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D007074:Immunoglobulin G / Q000378:metabolism; D007457:Iodine Radioisotopes; D009113:Muramidase / Q000378:metabolism; D010047:Ovalbumin / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D011817:Rabbits; D013745:Tetanus Toxoid / Q000276:immunology
chemical_list: D000906:Antibodies; D000888:Antibodies, Anti-Idiotypic; D002021:Buffers; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007457:Iodine Radioisotopes; D011506:Proteins; D013745:Tetanus Toxoid; D003101:Collodion; D010047:Ovalbumin; D009113:Muramidase