Sonics VCX750 User Manual download pdf (Page 23)

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.

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Sonics VCX750 User Manual Page 23