Cell disruption

As cell disruption (or homogenization ) method referred to in the biochemistry, in which cells to be destroyed in order for their content - proceed - organelles, proteins, DNA, mRNA, or other biomolecules.

Mechanical pulping processes

Physical digestion methods usually lead to a rise in temperature, so the temperature must be controlled and, if necessary, the disintegrated cooled in an ice bath or the digestion be carried out intermittently. Animal cells and plant cells need to be removed prior to pulping of unwanted tissues by being first crushed coarsely with scissors, scalpel or meat grinder. In addition, the tissue to be digested by multicellular organisms prior to homogenization in a mixer with rotating blades is crushed (or an Ultra- Turrax at low volume ).

• The Potter - Elvehjem procedure applies if the cell organelles are to be retained. Here, a piston which is tightly surrounded by a stationary vessel moves. By shear forces, the cells are destroyed.
• For micro-organisms, but also all other cells small volumes can be ground in a mortar with sand or Al2O3 or in a glass bead mill.
• By ultrasound, the cells are removed by cavitation constantly butted and sheared. Chromosomal DNA breaks preferably centrally due to the mechanical vibration. In all these processes much heat is developed in the treatment of larger volumes.
• For continuous or volume > 1000 ml, the sample can be forced under high pressure through a narrow valve ( Manton- Gaulin homogenizer ). Again, act cavitation. The digestion under pressure on a small scale takes place by means of French -press, which works according to the same principle as the Manton- Gaulin homogenizer.
• With the nitrogen Decompression can be quickly and gently unlock both animal and plant cells as well as bacteria susceptible regardless of the sample size in a simple way. Here in the cells according to the Henry's Law nitrogen is enriched at higher gas pressures. A sudden pressure relief leads to a burst of cell membranes.
• Repeated freezing and thawing the cells are destroyed by shear forces of the resulting ice crystals and perforation. By treatment with hypotonic buffer solutions, the lysis of the cells is enhanced. However, the combination of these methods does not lead to complete homogenization.

Non - mechanical pulping process

Non - mechanical pulping processes are applied to cells that are not easy to break ( for example, in yeast ). They are usually gentler than the mechanical method.

• In non- plant cells can be variants of the phenol -chloroform extraction, such as by Trizol extraction of the membrane lipids of the cell membrane of a decomposition are carried out, but in the presence of guanidinium thiocyanate is denaturing.
• In yeast autolysis is induced with toluene, which holes in the cell membrane and lysis with zymolyase enzyme destroys the cell wall glucan as Triton X-100 destroys the cell membrane.
• Gram - positive bacteria Peptidoglycanhülle is destroyed by treatment with lysozyme, then, the cell membrane is dissolved with Triton X-100.
• Gram -negative bacteria, the lipopolysaccharide of the outer cell membrane by treatment with EDTA, the Peptidoglycanhülle is destroyed by the treatment with lysozyme, and then the cell membrane may be destroyed by Triton X-100.
• Gram -negative bacteria, the membrane lipids may be hydrolyzed by alkaline lysis, this method is also a denaturing due to high pH.

Protein purification

Due to the disruption of the cells, the proteins get into a non-physiological environment and must be protected during protein purification from inactivation, denaturation and proteolysis. Since proteases are released during the digestion, fast work at low temperatures ( close to 0 ° C ) is necessary for higher yields.

Proteolysis

To avoid a degradation of the proteins protease inhibitors are added.

PH change

Due to the still partially running off metabolism, the pH may change. Therefore, to ensure adequate buffering. Optionally readjusting the pH by adding ammonia or tris (hydroxymethyl) aminomethane solution ( Tris) is necessary.

Ionic strength

An ionic strength of less than 0.05 M can lead to the formation of inclusion bodies and a low yield, since proteins attach to each other and to cell debris. Therefore, the digestion buffer solution should be 0.05 to 0.1 M sodium chloride or potassium chloride.

Hydrophobic effects

An aggregation of the proteins by van der Waals interactions and the formation of inclusion bodies because of hydrophobic effects can be obtained by adding non-ionic detergents such as Triton X -100 ( about 0.02 % w / v) Lubrol PX, or (about 0.006 % w / v ) can be reduced.

Thiol oxidation

The oxidation of thiol groups can be avoided to the buffer solution ( in mM concentrations digit ) by the addition of dithiothreitol (DTT), dithioerythritol (DTE) or 2 -mercaptoethanol. This is particularly necessary when cytosolic proteins since there prevails a reducing environment.

Heavy metal ions,

Heavy metal ions can react with functional groups of the protein. Divalent cations (Ca2 , Mg2 ) activate metalloproteases, therefore, ethylenediaminetetraacetic acid ( EDTA ) was added in mM concentrations, which forms complexes with metal ions.

DNA purification

Cell disruption by a variety of methods can be used for purification of DNA. To inactivate deoxyribonucleases EDTA is used as a chelator of its cofactor (magnesium ion ).