Chapter 7 : Cell Fractionation

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This chapter presents techniques for the fractionation of cellular components, including organelles and appendages, beginning with external surfaces and ending with internal components. It should be appreciated, however, that many of the techniques described in the chapter, from cell breakage to centrifugation theory, will have similar applications. The factors pertinent to cell fractionation, often dictating the approach needed, are the differences in wall structures and the bonding forces within walls (and associated surface materials) responsible for maintaining cell shape. Purification of the murein-outer membrane complex from the crude envelope fraction by density gradient separation in sucrose is described. Lipopolysaccharide (LPS) isolated by the phenol-water method preserves the reactivity of pseudomonad LPS with antibodies in Western immunoblots and is better in this regard than LPS isolated by the Darveau-Hancock method. In general, the murein portion of the cell wall is first removed to form protoplasts or spheroplasts, which are then lysed to provide either the plasma membrane or the plasma plus outer membranes. A structurally intact, intracytoplasmic membrane array may best be isolated by lysis of osmotically sensitive spheroplasts. The effect of lysis and physiological conditions on the nucleoid is discussed by Korch.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7

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Bacteria and Archaea
Plasma Membrane
Outer Membrane Proteins
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Isolation of cytoplasmic membranes by discontinuous sucrose gradient centrifugation. A membrane fraction from spheroplast lysate in 33% sucrose was loaded on 40 to 50% sucrose steps and centrifuged at 4°C for 48 h (110,000 × , max) in a Beckman SW27 swinging-bucket rotor. A thin section of the upper band revealed closed vesicles bounded by the double-track membrane, typical of cytoplasmic membranes. See reference 135 for further details.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Isolation of envelopes and related fractions from gram-negative bacteria.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Some commonly used methods to lyse bacteria and archaea to form osmotically sensitive cells in hypotonic solutions

Lysozyme susceptibility follows growth in medium containing penicillin G.

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7
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Buoyant densities of fractions isolated from prokaryotic cells

Citation: Koval S, Sprott G. 2007. Cell Fractionation, p 108-137. In Reddy C, Beveridge T, Breznak J, Marzluf G, Schmidt T, Snyder L (ed), Methods for General and Molecular Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817497.ch7

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