Removal of Cell Debris
The initial extract, produced by the disruption of cells and tissue, and referred to at this stage as a homogenate (or lysate), will invariably contain insoluble matter. For example, for mammalian tissue there will be incompletely homogenised connective and/or vascular tissue, and small fragments of non-homogenised tissue. This is most easily removed by filtering through a double layer of cheesecloth or by low speed (5000×g) centrifugation. Any fat floating on the surface can be removed by coarse filtration through glass wool or cheesecloth. However, the solution will still be cloudy with organelles and membrane fragments that are too small to be conveniently removed by filtration or low speed centrifugation. If necessary, they can be removed first by precipitation using materials such as celite (a diatomaceous earth that pro vides a large surface area to trap the particles), cell debris remover (a cellulose-based absorber), or any type of flocculants such as starch, gums, tannins or polyamines; the resultant precipitate being removed by centrifugation (typically about 20000×g for 45 min; or 40000×g for 30 min in an ultracentrifuge) or filtration.
Non-Protein Components
Even the cleared homogenate contains not only proteins, but also other molecules such as DNA, RNA, carbohydrate and lipid, as well as any number of small-molecular-weight metabolites. Small molecules tend to be removed later on during dialysis steps or steps that involve fractionation based on size (e.g. size-exclusion chromatography) and therefore are of little concern. However, specific attention has to be paid at this stage to macromolecules such as nucleic acids and polysaccharides. This is particularly true for bacterial extracts, which are particularly viscous owing to the presence of chromosomal DNA. Indeed microbial extracts can be extremely difficult to centrifuge to produce a supernatant extract. Some workers include DNase I in the extraction buffer to reduce viscosity, the small DNA fragments generated being removed at later dialysis/size-exclusion steps. Likewise, RNA can be removed by treatment with RNase. DNA and RNA can also be removed by precipitation with protamines – a mixture of small, highly basic (i.e. positively charged) proteins, whose natural role is to bind to DNA in the sperm head. Protamines are usually extracted from fish organs, which are obtained as a waste product at canning factories. These positively charged proteins bind to negatively charged phosphate groups on nucleic acids, thus masking them and rendering them insoluble. The addition of a solution of protamines to the homogenate therefore precipitates most of the DNA and RNA, which can subsequently be removed by centrifugation. An alternative is to use polyethyleneimine, a synthetic long-chain cationic polymer with molecular mass of about 24 kDa. This polymer also binds to the phosphate groups in nucleic acids, and is very effective, precipitating DNA and RNA almost instantly.
For bacterial extracts, carbohydrate capsular gum can also be a problem as this can interfere with protein precipitation methods. This is best removed by the use of lysozyme, which can digest capsular gum and is more efficient in the presence of detergent used to lyse the cells.
