Analytical isoelectric focusing in polymerizable thin layers containing sephadex

Isoelectric focusing (IEF) has, in recent years, become an extremely sensitive and widely employed tool for the detection of very small differences in the isoelectric points (PI) of various substances, including proteins and RNAs [ 11. For comparison of multiple samples, analytical IEF in thin-layers of polyacrylamide has proved to be of considerable value, since the gel plates are easy to handle, the resolution of components is excellent [2] and the focused substances can be detected by a number of methods [l] . However, very large molecules cannot be focused in these gels either because they do not enter the gel or because excessively long running times are required to reach equilibrium. These drawbacks can be overcome by using Sephadex as anti-convective medium [3]. Focused proteins are identifiable by a paper print which is made of the Sephadex thin-layer after the focusing process. The paper is dried in an oven and then stained [3]. Enzymes can be detected by substrate impregnated papers [4]. We have developed a simpler technique in which substances are focused in a Sephadex layer in the presence of acrylamide monomer and a cross-linking agent. After IEF, the whole layer is polymerized by spraying a solution containing polymerization initiators in a concentrated buffer onto the gel. Focused material can then be identified by standard methods. The advantages of this new approach are the possibility of focusing very large molecules or even viruses on thin-layers combined with the ease of handling a polymerized gel and the use of a variety of established methods to detect the focused substances. The disadvantages of IEF in sucrose gradients (bad resolution