The initial step in purifying subcellular structures is to rupture the plasma membrane and the cell wall, if present. First, the cells are suspended in a solution of appropriate pH and salt content, usually isotonic sucrose (0.25 M) or a combination of salts similar in composition to those in the cell's interior. Many cells can then be broken by stirring the cell suspension in a high-speed blender or by exposing it to ultrahigh-frequency sound (sonication). Plasma membranes can also be sheared by special pressurized tissue homogeniz-
▲ EXPERIMENTAL FIGURE 5-36 Differential centrifugation is a common first step in fractionating a cell homogenate.
The homogenate resulting from disrupting cells Is usually filtered to remove unbroken cells and then centrifuged at a fairly low speed to selectively pellet the nucleus—the largest organelle. The undeposited material (the supernatant) is next centrifuged at a higher speed to sediment the mitochondria, chloroplasts, lysosomes, and peroxisomes. Subsequent centrifugation in the ers in which the cells are forced through a very narrow space between the plunger and the vessel wall. As noted earlier, water flows into cells when they are placed in a hypotonic solution (see Figure 5-18). This osmotic flow causes cells to swell, weakening the plasma membrane and facilitating its rupture. Generally, the cell solution is kept at 0 °C to best preserve enzymes and other constituents after their release from the stabilizing forces of the cell.
Disrupting the cell produces a mix of suspended cellular components, the homogenate, from which the desired organelles can be retrieved. Homogenization of the cell and dilution of the cytosol cause the depolymerization of actin microfilaments and microtubules, releasing their monomeric subunits, and shear intermediate filaments into short fragments. Thus other procedures, described in Chapters 19 and 20, are used to study these important constituents. Because rat liver contains an abundance of a single cell type, this tissue has been used in many classic studies of cell organelles. However, the same isolation principles apply to virtually all cells and tissues, and modifications of these cell-fractionation techniques can be used to separate and purify any desired components.
ultracentrifuge at 100,000g for 60 minutes results in deposition of the plasma membrane, fragments of the endoplasmic reticulum, and large polyribosomes. The recovery of ribosomal subunits, small polyribosomes, and particles such as complexes of enzymes requires additional centrifugation at still higher speeds. Only the cytosol—the soluble aqueous part of the cytoplasm—remains in the supernatant after centrifugation at 300,000g for 2 hours.
Filter homogenate to remove clumps of unbroken cells, connective tissue, etc.
Mitochondria, chloroplasts, lysosomes, and peroxisomes
Was this article helpful?
Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.