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An electroimmunoblot technique was used to detect various forms of myelin basic protein (MBP) in brain homogenates of 14 vertebrate species. Three antibodies were used to probe the immunoblots: a monoclonal anti-human MPB reacting with an antigenic determinant located at amino acid residues 131 to 136; a polyclonal anti-human MBP and a polyclonal anti-chicken MBP. Because no processing of the tissue is required prior to electrophoresis, in vitro artifacts are minimized. The 18.5 K form of MBP was present in all species except the shark. A 21.5 K MBP was observed in ovine, bovine, pig, rabbit, mouse, rat, monkey, but not in human, guinea pig, shark, toad and marsupial brains. A variant with a molecular weight between 17 K and 18 K was found in mouse, rat, bovine, human, monkey, pig, and chicken brains, and was the sole component in the shark brain. Marsupial brains had five or six forms of MBP between 14.5 K and 18.5 K.

1. The complete amino acid sequence of the smaller basic protein from rat brain myelin was determined. This protein differs from myelin basic proteins of other species in having a deletion of a polypeptide of 40 amino acid residues from the centre of the molecule. 2. A detailed comparison is made of the constant and variable regions in a group of myelin basic proteins from six species. 3. An arginine residue in the rat protein was found to be partially methylated. The ratio of methylated to unmethylated arginine at this position differed from that found for the human basic protein. 4. Three tryptic peptides were isolated in more than one form. The differences between the two forms of each peptide are discussed in relation to the electrophoretic heterogeneity of myelin basic proteins, which is known to occur at alkaline pH values. 5. Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50029 at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1973) 131, 5.

While the techniques of ion-exchange chromatography, on derivatives of cellulose(l) and dextran gel(2), and gel fitration chromatography (3-7) have proved valuable for the fractionation of serologically active proteins, they have not been used previously in direct combination with immunodiffusion. Recently proteins have been separated on a micro-scale by thin layer chromatography on dextran gels(8-10) and by paper chromatography on anion-exchange cellulose (11). This report describes the combination of chromatography and immunodiffusion on a microscope slide to separate and characterize serologically active compounds in a manner analogous to immunoelectrophoresis (12).Materials and methods.The cross-linked dextran gels, diethylaminoethyl-Sephadex (DEAE) A-50 medium grade, carboxymethyl-Sephadex (CM) C-50 medium grade and Sephadex G-100 (G-100) 140-400 mesh, were prepared for use according to the manufacturer's instructions.§ In the experiments illustrated in Fig. 1, DEAE was equilibrated with sodium phosphate buffer (pH 5.8, 0.05 M), CM with sodium acetate buffer (pH 5.5, 0.1 M), and G-100 with sodium phosphate buffer (pH 8.0, 0.1 M in 0.5 M sodium chloride). For diffusion from CM and G-100 columns a 2% agar gel (Difco, Bacto-Agar) with 1% sodium azide in veronal-HCl buffer (pH 7.2, 0.2 M) was used, whereas, for diffusion from the DEAE columns phosphate buffer (pH 5.8, 0.1 M in 0.5 M sodium chloride) was necessary. Molten agar (2 ml) was pipetted onto a standard microscope slide which had been previously coated with 0.1% agar. After the agar had solidified a longitudinal halft was removed and a trough 1.4 mm wide × 7.2 cm long was cut 5 mm from the center of the slide in the remainder of the agar. Dextran gel columns of reproducible dimensions were prepard on the vacant half of the slide.