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evaporation with a stream of nitrogen and subsequently dialyzed to buffer III or whatever SDS system is desired for the fractionation by gel electrophoresis. The individual NHC protein bands can then be used to obtain samples for further chemical characterization as discussed above. Acknowledgments

The author is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. Work from this laboratory and writing were supported by the Jane Coffin Childs Fund and National Science Foundation Grant GB 34160 to Dr. Leroy E. Hood.

[12] Methods for Isolation and Characterization of Chromosomal Nonhistone Proteins Fractionation of Chromatin on Hydroxyapatite and Characterization of the Nonhistone Proteins by Ion Exchange Chromatography and Polyacrylamide Gel Electrophoresis

B y A. J. MACGILLIVRAY,D. RICKWOOD, A. CAMERON, D. CARROLL, C. J. INGLES, R. J. KRAUZE, and J. PAUL There are at present numerous methods available for the separation of the nonhistone proteins from other constituents of chromatin. As we have described elsewhere, 1 many of these procedures are multistep, suffer from incomplete extraction of the nonhistone proteins, and expose the proteins to extreme conditions of pH and solvent environment. Since there is a requirement for methodology which can avoid these defects, we have been interested in utilizing the combination of salt and urea (e.g., 2--3 M NaC1 and 5-7 M urea) which has been used as a solvent both for the dissociation and solubilization of chromatin and its specific reconstruction from previously isolated components. -~,3 The following methodology is based on the ability of hydroxyapatite to act as an ion exchange medium for Inacromolecules in the presence of high ionic strength and urea 4,5 and consists of a single column procedure, yielding high recoveries of chromatin constituents. 1A. J. MacGillivray in "Subnuclear Components" (G. D. Birnie, ed.). Butterworths, London, in press. 2I. Bekhor, G. M. Kung, and J. Bonner, J. Mol. Biol. 39, 351 (1969). R. S. Gilmour and J. Paul, J. Mol. Biol. 40, 137 (1969). 4G. Bernardi and T. Kawasaki, Biochim. Biophys. Acta 160, 301 (1968). 5G. Bernardi, M. G. Giro, and C. Galliard, Biochim. Biophys. Acta 278, 409 (1972).

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PREPARATION OF CHROMATIN NONHISTONE PROTEINS

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Methods

Chemicals As far as possible these were of Analar grade and were largely obtained from British Drug Houses Ltd., Poole, Dorset. "Analar" grade urea was routinely prepared as an 8 M stock solution, filtered through Whatman No. 1 filter paper, and deionized immediately before use by passage through a column of AG 501-X8 (D) mixed bed resin (Bio-Rad Laboratories Ltd., St. Albans, Herts.). Calcium chloride and disodium hydrogen phosphate for preparing hydroxyapatite were purchased from Merck Chemicals Ltd., Darmstadt, Germany. QAE-Sephadex was purchased from Pharmacia (G.B.) Ltd., London. Acrylamide, N,N'methylenebisacrylamide, and N,N,N',N'-tetramethylethylenediamine were purchased from Kodak Ltd., London. The acrylamide and N,N'-methylenebisacrylamidc were recrystallized before use. Ampholines (pH range 3-10) were purchased from LKB Instruments Ltd., South Croydon. Standard marker proteins, bovine serum albumin, ovalbumin, chymotrypsinogen, trypsin, lysozyme, and ribonuclease were purchased from the Sigma Chemical Co. Ltd., London. Coomassie Brilliant Blue R and Carbowax (MW 15,000-20,000) were purchased from G. T. Gurr, High Wycombe, Bucks.

Preparation o] Nuclei Except where stated all procedures are carried out at 0-4 ° . Male mice (12 weeks old) were used in all experiments; mouse organs are excised, placed in ice-cold 0.25 M sucrose, and used immediately for the preparation of nuclei. Nuclei are prepared by a modification of the method of Widnell and Tata2 The tissue is homogenized in three volumes of 0.25 M sucrose-3 mM CaC12 in a glass-Teflon Potter-Elvehjem type homogenizer using eight up and down strokes at 2000 rpm. After filtration through four layers of gauze, the homogenate is centrifuged at 1000 g for 10 minutes. The pellet is then homogenized in 2.4 M sucrose-3 mM CaC12 and centrifuged at 40,000 g for 1 hour. The supernatant is discarded, and the pelleted nuclei are washed in 0.25 M sucrose-3 mM CaCl_~ and collected by centrifugation at 1000 g for 10 minutes. The nuclei are suspended in the same medium containing 1% (v/v) Triton X-100, which removes about 90% of the inner and outer nuclear membranes, 7 as judged by elecC. C. Widnell and J. R. Tata, Biochim. Biophys. Acta 123, 478 (1966). 7 A. D. Barton, W. E. Kisieleski, F. Wasserman, and F. Mackevicius, Z. Zell]orsch. Mikrosk. Anat. 115~ 299 (1971).

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iron microscopy. The Triton X-100 is removed by washing the nuclei twice in 0.25 M sucrose-3 mM CaC12. The nuclei are then stored as a pellet at --20°C.

Preparation o] Chromatin Chromatin is prepared from the purified nuclei by homogenizing the nuclei in a tight-fitting glass-Teflon homogenizer in 50 volumes of 0.14 M NaC1-50 mM Tris • HC1, pH 7.4 with eight up-and-down strokes at 2000 rpm. The suspended chromatin is stirred for 20 minutes and pelleted by centrifugation at 15,000 g for 10 minutes. The chromatin is extracted twice more with the same volume of saline-Tris buffer and pelleted as before.

Separation o/Chromatin Components The ehromatin is homogenized in a glass-Teflon homogenizer in 30 volumes of 1 mM sodium phosphate, pH 6.8-2 M NaC1-5 M urea. The solubilized chromatin is centrifuged at 15,000 g for 10 minutes and the chromatin is reextracted with half the original amount of the same solution. The pooled ehromatin extracts are then sonicated for 15 seconds using an MSE Ultrasonic Power Unit at a setting of 1.5 A and centrifuged at 10,000 g to remove any small residual material. Hydroxyapatite, prepared by the method of Bernardi, 8 is fined several times in 1 mM sodium phosphate, pH 6.8-2 M NaC1-5 M urea by suspension and decantation. Columns of hydroxyapatite (25 em X 1.6 cm) are packed and equilibrated with the above solution at room temperature and run at 10-12 ml per hour. The hydroxyapatite column is loaded with chromatin (up to 20 mg of DNA) dissociated in 1 mM sodium phosphate, pH 6.8-2 M NaC1-5 M urea ; larger amounts of chromatin require proportionally larger columns. The chromatin fractionation used is outlined in Fig. 1. Some of the chromatin proteins are not retained by hydroxyapatire; this fraction (H1) contains no nucleic acid (Fig. 1), and analysis of these proteins by sodium dodecyl sulfate gels (Fig. 2) and amino acid analysis° shows that it consists of essentially pure total cell histone. After elution of the unretained material with equilibrium buffer, the bulk of the nonhistone proteins (fraction H2), together with a small amount of RNA (approximately 4% of the protein), is eluted with 50 mM sodium phosphate, pH 6.8 in salt-urea. The remainder of the nonhistone proteins (fraction H3) is eluted, together with an equal amount of RNA, by 200 See this series, Vol. 21 [3]. 9 A. J. MacGillivray, A. Cameron, R. J. Krauze, D. Rickwood, and J. Paul, Biochim. Biophys. Acta 277, 384 (1972).

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Methods for isolation and characterization of chromosomal nonhistone proteins. Fractionation of chromatin on hydroxyapatite and characterization of the nonhistone proteins by ion exchange chromatography and polyacrylamide gel electrophoresis.

160 THE CELL NUCLEUS AND CHROMATIN P R O TEIN S [12] evaporation with a stream of nitrogen and subsequently dialyzed to buffer III or whatever SDS...
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