The method of STEWARD, LYNDON, and BARBER (1965) has been used, with some modifications, for the electrophoretic separation in acrylamide gel columns of soluble proteins from different root sections of pea seedlings. The influence of several experimental variables on the effectiveness of separation was studied. The results deviate from results already in the literature.Different catalyzers for gel polymerisation (β-dimethyl amino-propionitrile, N,N,N′,N′-tetramethylethylenediamine) have a small but detectable influence upon the separation. Fresh staining solutions (amido black 10 b) improve separations compared with repeatedly used ones, probably due to faster protein denaturation. Destaining of columns in the direction of protein movement renders better results than destaining in the opposite direction, since during destaining the protein bands shift slightly in the direction of destaining. Comparison of columns of the same proteins destained in each direction is useful.Dialysis against Tris-glycine-buffer (pH 8.3) prior to separation diminished the number of protein bands, to different extents depending upon the developmental stage of the root sections. The protein bands of older root segments changed the least. Protein solutions dialyzed against double distilled water and returned to buffer showed fewer bands which was probably the result of denaturation. Different storage times for frozen protein solutions (-20 °C) as well as conditions during polymerisation of the acrylamide gel had effects on protein separation. In all cases proteins from older segments were more stable than those from younger segments.Comparison of our results with the literature shows that the quality of protein separation by disc electrophoresis in acrylamide is critically dependent upon methods of isolation and concentration of protein solutions. The upper limit of efficiency of the method seems to be about 30 protein bands at present.

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