Complete separation of the triplet components of neurofilament by DE-52 column chromatography depends upon urea concentration

In the separation of the triplet components of neurofilament (P 200, P 160, and P 68) by DE-52 column chromatography in the presence of urea, it was revealed that the efficiency of separation depended upon urea concentration. When chromatography was performed in the presence of 8 m urea and a linearly increasing sodium phosphate concentration from 10 to 400 mm at pH 6.8, P 160 and P 68 were eluted in the same peak, although P 200 was eluted faster. P 160 and P 68 were partially separated with 6 m urea, and completely separated with 4 m urea. But, under these conditions, P 200 was eluted at the same position as contaminated glial fibrillary acidic protein (GFA). From these results, two methods were recommended for the complete separation of the triplet components of neurofilament and GFA by DE-52 column chromatography. In one method, chromatography was performed in the presence of 8 m urea at first, and then P 160 and P 68 were separated in the presence of 4 m urea. In the other method, chromatography was performed with a linearly decreasing urea concentration from 8 to 0 m.