A large-scale procedure for isolation of nucleoli from Walker tumors.

A method is presented for isolation of 2-10 gm of highly puri fied nucleoli from Walker tumors. Initially, 0.5-1 kg of tumor was minced in a specially devised stainless steel mincer. The minced tissue was then homogenized in a continuous tissue homogenizer in 0.25 Msucrose containing 3.3 HIMCaClj. The nuclear preparation, obtained by continuous flow centrifugation, was subjected to sonic oscillation with a sonifier for periods of 1-2 min. The preparation was layered over 0.88 M sucrose and centrifuged for 20 min at 900 X g in a centrifuge with a large capac ity to provide the nucleolar pellet. Electron microscopic and chemical analyses indicated that the preparation was highly purified and the nucleoli were morphologically similar to those in the tumor cells. The yield of nucleoli/kg of tissue was approxi mately 7 X 10l°, i.e., 4.3 ml of packed nucleoli. Sucrose gradient