ISOLATION AND COMPOSITION OF NUCLEI AND NUCLEOLI 105 



95 volts a.c; or it can be run through the colloid mill which is operated at medium 

 speed (10,000 r.p.m.) with a clearance of about 1.5-thousandths of an inch. After the 

 final blending there will be considerable foam which tends to entrap fine fiber as it 

 rises to the top. By pipetting the homogenate from beneath the foam, fiber may be 

 conveniently removed. The temperature must be kept as close to 0° as possible, and 

 the addition of ice from time to time during the blending is desirable if the Waring 

 Blendor is used for the final homogenization. If the colloid mill is used, a mixture of 

 ice and water is placed in the funnel, and ice-water is used to wash the last of the liver 

 homogenate through the mill, since the mill rotor continues to spin for some time after 

 the current has been turned off and the mill must not be run dry. It is convenient to 

 have two persons operating the colloid mill and two passages of the homogenate 

 through the mill are sufficient. 



The isolation of the nuclei from the homogenate by differential centrifugation has 

 been adequately described in the original reference.' 



If nucleic acids or lipids only are to be studied, it may be permissible to isolate the 

 nuclei in strong citric acid which facilitates the rapid preparation of very clean nuclei 

 from a wide variety of tissues and even from tumor tissues where milder methods 

 usually fail. Such methods are described in references 10, 11, 12, and 13 and similar 

 methods have also been published by Barnum et al.,^^ by Mirsky and Pollister,'^ and 

 by Frazer and Davidson.** 



The method of isolation of nuclei at pH 5.9 to 6.0 is similar to that just described 

 except that more dilute citric acid is used. In the original method* chopped frozen 

 liver was added to ice-cold very dilute citric acid in the Waring Blendor. In a subse- 

 quent modification,''^ the liver was blended for a very short time in ice-water in the 

 Blendor and citric acid was then added dropwise. For reasons outlined elsewhere, we 

 now believe it is preferable to add the liver to the dilute citric acid in the Blendor, and 

 after a short mixing (of about 1 min.) to filter as described above. Homogenization is 

 then completed by operating the Waring Blendor at 95 volts a.c. for 7.5 min. for 50 g. 

 of liver (15 min. for 100 g.); or the colloid mill may be used as described for nuclei 

 prepared at pH 4.0. If the liver is to be added to the citric acid solution as now recom- 

 mended, 50 g. of frozen liver is added to 200 ml. of ice-cold water containing 5.6 ml. 

 0.1 M citric acid. Since this amount of acid will lower the pH to 5.9 to 6.0, the pro- 

 cedure is safer than the use of a pH range of 6.0 to 6.2 as originally recommended, since 

 fewer nuclei will be disintegrated. A little more acid may be added as required to ad- 

 just the pH to 5.9; it should not be allowed to rise above 6.0. 



The centrifuging schedule is similar to that used for preparing nuclei at pH 4.0, 

 and the final washings are carried out in the same way. The nuclei may be transferred 

 to a smaller centrifuge tube for the last two or three washings. 



(2) Methods Involving Solutions of Gum Arabic in Water. The isolation of nuclei at 

 pH 5.9 to 6.0 is greatly facilitated by the addition of ice-cold 5% gum arabic solution 

 (previously adjusted to pH 6.0) to the homogenate just before the first centrifuga- 

 tion. ^^ Enough of the gum arabic solution is added to make the final concentration of 

 gum 1%. Subsequent washings are carried out with 1% gum arabic solution adjusted 

 to pH 6.0. The addition of gum arabic also tends to lessen the adsorption of frag- 

 mented mitochondria by the nuclei. 



*^ C. P. Barnum, C. W. Nash, E. Jennings, O. Nygaard, and H. Vermund, Arch. Bio- 



c/iem. 26, 376 (1950). 

 " S. C. Frazer and J. N. Davidson, Exptl. Cell Research 4, 316 (1953). 

 " A. L. Dounce, Ann. N. Y. Acad. Sci. 50, 982 (1950). 

 28 A. L. Dounce and M. Litt, Federation Proc. 11, 203 (1952). 



