By Stein, Gary.; Stein, Janet.; Kleinsmith, Lewis J
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Extra resources for Chromatin and chromosomal protein research : III
G . D. BIRNIE Buoyant Density-Gradient Centrifugation Although high-molecular-weight chromatin can be sedimented to its isopycnic point quite satisfactorily and rapidly in preformed 20-60% (w/v) metrizamide gradients, this method is not generally suitable for sheared chromatin because of the very long time of centrifugation required to ensure that the equilibrium positions have been reached by all species of particles. Moreover, the amount of chromatin which can be fractionated on a preformed gradient is limited.
CHROMATIN FRACTIONATION M METRIZAMIDE 39 REFERENCES 1. Slayter, H. , Shih, T. (1972). ,Adler, A. , and Fasman, G. , Biochemistry 11, 3044 2. , and McCarthy, B. , Biochemistry 14, 1366 (1975). 3. Ilyin, Y. , Varshavsky, A. , Mickelsaar, U. , and Georgiev, G. , Eur. J. Eiochem. 22, 235 (1971). 4. Varshavsky, A. J. and Georgiev, G. , Biochim. Eiophys. Acta 281, 669 (1972). 5. Varshavsky, A. , and Georgiev, G. , Mol. Eiol. Rep. 1, 143 (1973). 6. Varshavsky, A. , Jlyin, Y. , and Georgiev, G. , Nature (London) 250, 602 (1974).
Digestion of nuclei was carried out on suspensions of 3. 25Msucrose and resuspended in the same buffer at a DNA concentration of 500 pg/ml. The kinetics of DNA digestion following the addition of nuclease were assayed by measuring the amount of A 260 absorbing material soluble in 1 M NaC1-1 M HC10,. RNA contributed less than 5% of the total A,,, of purified nuclei as determined either by alkali digestion or treatment with pancreatic ribonuclease. Nuclease reactions were terminated by the addition of NaEDTA to 5 mM.
Chromatin and chromosomal protein research : III by Stein, Gary.; Stein, Janet.; Kleinsmith, Lewis J