The Use of Isoelectric Focusing in the Separation and Characterization of Nuclear Nonhistone Proteins

MacGillivray, A.J.

doi:10.1007/978-1-4613-4181-9_6

Biological and Biomedical Applications of Isoelectric Focusing 1977

DOI: 10.1007/978-1-4613-4181-9_6

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The Use of Isoelectric Focusing in the Separation and Characterization of Nuclear Nonhistone Proteins

A.J. MacGillivray

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“…Further increase in the phosphate concentration merely eluted the DNA from the hydroxyapatite. Recently we have found that solutions of high concentrations of guanidinium hydrochloride to be an efficient solvent for non-histone proteins [30,31]. This communication describes the adaption of our previous procedures to include the use of guanidinium hydrochloride in order to obtain a more efficient recovery of nonhistone proteins.…”

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Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Rickwood

MacGillivray

1975

Eur J Biochem

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The effect of the dissociation medium on the fractionation of chromatin on hydroxyapatite has been studied. Optimal separations of the histones and non-histone protein are only achieved when columns are run in buffers containing high concentrations of sodium ions. We have modified our previously published method such that the chromosomal proteins can be recovered in virtually quantitative yields. Each of the hydroxyapatite fractions has been analysed with respect to nucleic acid content and the proteins have been analysed by two-dimensional gel electrophoresis.Recent evidence suggests that the non-histone proteins of chromatin play an important role in the control of transcription (see review by MacGillivray and Rickwood [l] [6] after the chromatin has been extracted with dilute acid to remove the basic histones. Alternatively, chromatin has been dissociated in sodium dodecyl sulphate [7] or in various salt solutions [8-201 so that after removal of the DNA by centrifugation [7][8][9]12,13,16,17] gel filtration [9-11,18, 191 or precipitation [14,15,20], the chromatin proteins have been obtained as such [I41 or separated from the histones by ion-exchange chromatography [8,[10][11][12][13][15][16][17][18][19][20] or by electrophoresis [7]. Other workers have used variations or combinations of some of these techniques [21-241. However, some of these methods subject the chromatin to extremes of pH and solvent environment which can be destructive to both proteins [25] and DNA [5,19], while others produce selected fractions [2,3,15] or yield low recoveries of non-histone proteins [ l l , 16,18,19]. Our approach has been to use non-rigorous conditions which yet give good yields of non-histone proteins. In our procedure chromatin is dissociated in 2 M NaCl-5 M urea and applied to a column of hydroxyapatite. This ion-exchange medium is not affected by the presence of high concentrations of urea and the phosphate sites which would otherwise bind basic molecules such as histones are effectively blocked by the sodium ions [26,27]. The acidic proteins bind to the Ca2+ sites of the column and are eluted by increasing the phosphate concentration [26]. Thus in the case of chromatin, we found that the histones are not retained and can be recovered in almost quantitative yield, but only 60%-70% of the adsorbed nonhistone proteins could be recovered using phosphate concentrations up to 0.2 M [28,29]. Further increase in the phosphate concentration merely eluted the DNA from the hydroxyapatite. Recently we have found that solutions of high concentrations of guanidinium hydrochloride to be an efficient solvent for non-histone proteins [30,31]. This communication describes the adaption of our previous procedures to include the use of guanidinium hydrochloride in order to obtain a more efficient recovery of nonhistone proteins. MATERIALS AND METHODS ChemicalsAs far as possible these were of AnalaR grade and were largely obtained from British Drug Houses Ltd (Poole, Dorset, U.K.). AnalaR-grade urea was routinely prepared as an 8 M sto...

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Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Rickwood

MacGillivray

1975

Eur J Biochem

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The Use of Isoelectric Focusing in the Separation and Characterization of Nuclear Nonhistone Proteins

Cited by 1 publication

References 78 publications

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

Improved Techniques for the Fractionation of Non-Histone Proteins of Chromatin on Hydroxyapatite

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