Press-blotting on gelatin-coated nitrocellulose membranes. A method for sensitive quantitative immunodetection of peptides after gel isoelectric focusing

Abstract

A method is presented for the fixation of peptides in nitrocellulose membranes after isoelectric focusing on thin polyacrylamide gels. Focusing gels are covered with gelatin-coated nitrocellulose membrane. Using glutaraldehyde, focused peptides are covalently fixed onto this membrane. Fixed peptides are stained using the peroxidase-anti-peroxidase method and the immunoreaction is quantified by rendering the membrane transparent and measuring the optical density of the precipitated chromogen in each band. The effect of pore size and gelatin content of the membrane, glutaraldehyde concentration and fixation time on fixation efficiency and immunostaining has been investigated. Gelatin coating considerably increases the efficiency of glutaraldehyde fixation of peptides and greatly enhances antibody-binding. Consequently, sensitive quantitative immunodetection is possible and, depending on the antiserum, peptides are readily detected in quantities down to 10 pg.