Protein antigen purification by preparative protein blotting

Abstract

AbstractRecent reports have described procedures for recovering proteins from nitrocellulose membrane blots, either by dissolution of the membrane with acetone or dimethyl‐sulfoxide (DMSO) or by specific desorption of the immobilized proteins with acetonitrile or pyridine. In the current review, various procedures have been evaluated for the recovery of protein antigens from blots on nitrocellulose, Zeta Probe nylon, Immobilon, or glass fiber membranes. The results of this survey indicate that elution with appropriate concentrations of acetonitrile affects equally efficient desorption of proteins bound to nitrocellulose, Zeta Probe, or Immobilon membranes, while formic acid was effective in eluting proteins from glass fiber or Zeta Probe membranes. Although nitrocellulose blots could be dissolved in acetone or DMSO for antigen production, other membrane matrices were resistant to dissolution by organic solvents, except for Immobilon in DMSO. The use of volatile solvents permits the reconstitution of the isolated lyophilized protein into the buffer of choice with retained protein structure or biological activities (e. g. antigenicity). Nitrocellulose appeared to be most versatile for preparative protein blotting because it displays a protein binding capacity equivalent to other membranes, effectively retains a variety of large and small molecular weight proteins, and is compatible with reversible protein staining by Ponceau S.