Sonics VC750 (Serial No. "Y through "AB")" - Manual (Page 21)

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SECTION IV -
OPERATING SUGGESTIONS AND TECHNIQUES
DISRUPTING CELLS
The disruption of cells is an important method in the field of proteomics and in the
isolation and preparation of intracellular products. Isolation of subcellular fractions
and concentration of proteins allow for more efficient identification and study of
proteins of interest. From research levels through to production, many areas of
biotechnology, particularly recombinant technology, necessitate the use of ultrasonics
for cell disruption. Cell disruption focuses on obtaining the desired product from
within the cell, and it is the cell wall that must be disrupted to allow access to the
contents of the cell.
All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier
separating cell contents from the extracellular environment. Lipids constituting the
plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that
associates spontaneously to form a closed bimolecular sheet. Membrane proteins are
embedded in the lipid bilayer, held in place by one or more domains spanning the
hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of
the bilayer through interactions with integral membrane protein or with polar lipid head
groups. The nature of the lipid and protein content varies with cell type.
In animal cells, the plasma membrane is the only barrier separating cell contents from
the environment, but in plants the plasma membrane is also surrounded by a rigid cell
wall. Plant cell walls consist of multiple layers of cellulose. These types of extracellular
barrier confer shape and rigidity to the cells. Plant cell walls are particularly tough,
making them very difficult to disrupt mechanically or chemically. The lack of an
extracellular wall in animal cells make them relatively easy to lyse.
Soft, fresh plant tissue can often be disrupted by sonicating in a lysis buffer. Other
plant tissues, like pine needles, need to be ground dry, without liquid nitrogen. Some
hard, woody plant materials require freezing and grinding in liquid nitrogen prior to
being ultrasonically processed. Plant cell suspension cultures and calluses can be
lysed by sonication in a lysis buffer for 30 seconds to 2 minutes. The diversity of
plants and plant tissue make it impossible to give a single recommendation for all
samples. However, one should be aware that most plant tissues typically contain
polysaccharides and polyphenols that can coprecipitate with RNA and inhibit
downstream assays. Treating a plant tissue lysate with polyvinylpyrrolidone (PVP) will
precipitate such problematic components from the lysate before the actual RNA
isolation is carried out.