Amphoteric, isoelectric agarose membranes, as devised by Martin and Hampson [Martin, A.J.P. and Hampson, F. (1978) J. Chromatogr. 159, 101-110], are found unsuitable for blocking electroendosmosis in multi-compartment electrolysers during preparative isoelectric focusing, due to the poor and highly unpredictable incorporation of carboxyls and amino groups on the polysaccharide moiety. New, polyacrylamide-based membranes are described, containing as buffers and titrants the Immobiline chemicals used to produce immobilized pH gradients. These new membranes are supported on both faces by a non-woven polypropylene cloth, a material exhibiting minimal adsorption properties for proteins. Due to the extensively developed Immobiline technology, membranes with highly predictable isoelectric points, well-defined buffering capacity and conductivity can be synthesized at any pH value along the pH 3-10 scale. They are effective in blocking electroendosmosis even when the delta pH on either side of the membrane is as high as 1.5 pH unit.