Nuclear proteins. VI. Fractionation of chromosomal non-histone proteins using hydrophobic chromatography

Mouse liver non-histone proteins, isolated by hydroxyapatite chromatography, were fractionated by hydrophobic chromatography using omega-amino-decyl-agarose omega-amino butyl-agarose, decyl-agarose, butyl-agarose, phenyl-Sepharose, and CPAD-Sepharose. Two column loading techniques were used. In the 0.35 M NaCl technique, the proteins were dialized into 0.35 M NaCl, applied to the column and initially eluted with 0.35 M NaCl. In the 40% (NH4)2SO4 technique, the non-histone proteins were mixed with the hydrophobic agarose, dialized against 40% (NH4)2SO4, and initially eluted with 40% (NH4)2SO4. In both cases the columns were subsequently eluted with 10 mM Tris-HCl, pH 7.5, 0.35, 1.0 and 5.0 M LiBr, and finally with 1% sodium dodecyl sulfate. The 0.35 M NaCl technique, using decyl-agarose and phenyl-Sepharose, resulted in a single step marked enrichment of the major hnRNA proteins (1 M LiBr fraction). The 40% (NH4)2SO4 technique resulted in a single step isolation of a pair of 15-20 000 dalton polypeptides.