By Becker N.G., Salim A., Kelman C.W.
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In the isolation medium. 3. 2% Digltonin in isolation medium: Prepare this by dissolving the digltonin in the medium (without added BSA) with heating; then add the BSA. 4. Loose-fitting Dounce homogenizer. 5. Sonicator (see Note 13). 6. , polycarbonate) tubes for fixed-angle rotors of high-speed and ultracentrifuges. Volumes will depend on amount of material. 4. Outer Mitochondrial Membranes 1. 02% BSA. 2. , polycarbonate) tubes (about 50 mL) for fixed-angle rotors for low-speed and high-speed centrifuges.
The number of strokes needed will depend on a number of parameters (the size, clearance, thrust, and speed of the pestle and the size of the liver fragments). 5. Tissue, such as muscle, will need a blender type of homogenizer rather than a ltqutd shear one. 6. The homogemzation methodology for tissue culture cells needs to be worked out for mdrvtdual cell types (see Chapter 9). The mclusion of both monovalent and divalent cations in the homogemzation medium usually makes the rupture of cultured animal cells by standard liquid shear techmques (Potter-Elvehjem or Dounce) very difficult.
Resuspend the punfied nuclei m Buffer 1 (approx 5 x 10*/r&); add l/10 vol of each of the enzyme solutions, and incubate for 1 h. Isolation of Nuclei 25 3. Centrifuge at 1OOOgfor 10 mm, and resuspend in the same volume of Buffer 2. 4. 6M and P-mercaptoethanol to 1% (v/v). 5. After incubating for 15 mm, centrifuge at 2000g for 30 min. 6. 6M with respect to NaCl. 7. Repeat step 4, and resuspend the pellet of purified nuclear envelopes in a suitable buffer (see Notes 12-14). 5. 1. Using 1 mM NaHCO3 1.
Analysis of a potential trigger of an acute illness by Becker N.G., Salim A., Kelman C.W.