The erasable Western blot.
A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001665:Binding Sites; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000032:analysis / Q000276:immunology; D008892:Milk; D009685:Nuclear Envelope / Q000032:analysis / Q000276:immunology; D009757:Nylons; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D000906:Antibodies; D009757:Nylons; D010455:Peptides; D012967:Sodium Dodecyl Sulfate; D003101:Collodion