466 MECHANICAL SEPARATION OF CELLULAR COMPONENTS 



Lazarow Procedure for Separation and Isolation of 



Lipoprotein and Glycogen Particles from Guinea Pig Liver 



Prepare a suspension of fragmented liver cells in the manner de- 

 scribed for the preparation of mitochondria using a volume of saline 

 three times that of the liver (page 462) . Continue all operations in a 

 cold room. Clarify the suspension by spinning for two 10 min. periods 

 at 3000 R.P.M. in an 8 in. angle centrifuge, followed by 15 min. at 

 6000 R.P.M. Discard the precipitates after each centrifuging. Trans- 

 fer the final supernatant fluid to clean 10 ml. Lusteroid test tubes and 

 centrifuge at 12,000 R.P.M. for 30 min. The sediment consists of a 

 densely packed cake of the particulate glycogen, and a loosely 

 packed red precipitate which can easily be separated from the gly- 

 cogen by inverting the tube. The red precipitate is a mixture of the 

 glycogen and lipoprotein particles. After removing the red material, 

 wash the surface of the packed cake twice with physiological saline 

 soln. and then resuspend in saline allowing 30 min. for dispersion. 

 Centrifuge at 12,000 R.P.M. for 30 min. and discard the supernatant. 

 Repeat the resuspension and centrifugation four times. Dry the 

 final cake in a vacuum desiccator and a clean white powder con- 

 sisting mainly of glycogen is obtained. 



The red precipitate containing both lipoprotein and glycogen can 

 be freed of the latter by digestion with a purified diastase prepara- 

 tion. The separated fractions of both submicroscopic particulates 

 form transparent gelatinous pellets on centrifugation. 



Claude Procedure for Isolation of "Microsomes" 



All operations are carried out in a cold room at 0° and all solu- 

 tions employed are cooled to this temperature. 



Suspend the ground tissue mass in eight to ten times its weight 

 of either 0.005 M phosphate buffer, pB. 7.1, or 0.0002 A^ sodium 

 hydroxide and centrifuge 20 min. at 2400 times gravity. Subject the 

 supernatant fluid to high-speed centrifugation at about 18,000 times 

 gravity for 1 hr. Take up the sediment in a little water and centri- 

 fuge at the top speed for 3-5 min. to throw down the coarser 

 particles and then resuspend them and again centrifuge for 3-5 min. 

 Combine the supernatants from both short runs and repeat the 

 process of a 1 hr. run followed by two short runs two or three times. 

 In this fashion a concentration of particles ranging in diameter from 



