Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries.
We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.
D019747:Aldose-Ketose Isomerases; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification*; D000941:Antigens; D001923:Brain Chemistry; D002238:Carbohydrate Epimerases / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D003101:Collodion; D004247:DNA / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007106:Immune Sera / Q000302:isolation & purification; D007118:Immunoassay / Q000379:methods*; D007381:Intermediate Filament Proteins / Q000235:genetics; D016900:Neurofilament Proteins; D010209:Paper; D051381:Rats
D000906:Antibodies; D000941:Antigens; D007106:Immune Sera; D007381:Intermediate Filament Proteins; D016900:Neurofilament Proteins; D003101:Collodion; D004247:DNA; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase