Binding of antibodies and other proteins to nitrocellulose in acidic, basic, and chaotropic buffers.
This report compares the binding of proteins to nitrocellulose membranes in acidic buffers (pH 2 and 3) with binding in neutral buffer (pH 7), basic buffers (pH 12 and 13), 8 M urea (pH 2, 3, and 7), and 6 M guanidine hydrochloride (pH unadjusted). Initially, similar amounts of antibodies and other proteins bound to the nitrocellulose membrane in all of these buffers and solvents. However, the susceptibility of individual proteins to displacement (stripping) from the membrane by the milk blocking agent depended on both the pH and the type of buffer or solvent used to bind the proteins to the membrane. Most proteins that were bound to nitrocellulose in acidic buffers were relatively resistant to milk stripping compared to proteins bound in pH 7 buffer. After correction 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 using a milk block.
Hoffman
W L
WL
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
Jump
A A
AA
Kelly
P J
PJ
Ruggles
A O
AO
D000818:Animals; D000937:Antigen-Antibody Reactions; D001666:Binding Sites, Antibody; D015153:Blotting, Western / Q000379:methods*; D002021:Buffers; D003101:Collodion / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D008892:Milk; D011489:Protein Denaturation; D013745:Tetanus Toxoid / Q000276:immunology*
D002021:Buffers; D007074:Immunoglobulin G; D013745:Tetanus Toxoid; D003101:Collodion