Basic technology of biochemistry experiment: crushing technology
In addition to certain peptide hormones outside certain cells and certain proteins and enzymes, for the separation and purification of various biological macromolecules in intracellular or multicellular biological tissues, the cells and tissues need to be broken in advance so that the biological macromolecules are fully released Into the solution, there is no loss of biological activity. Different organisms or tissues in different parts of the same organism have different difficulty in cell disruption, and the methods used are different. For example, the cell membrane of animal organs is fragile and easily broken. Plants and microorganisms have strong Cell walls composed of cellulose and hemicellulose require special cell disruption methods.
1. Mechanical crushing
(1) Grinding
Grinding is to place the shredded animal tissue in a mortar or homogenizer, add a small amount of quartz sand to grind or homogenize, and then the animal cells can be broken. This method is relatively gentle and suitable for laboratory use. It can be ground by electric grinder in industrial production. This method can also be used to break up bacteria and plant tissue cells.
(2) homogenate
Homogenization This is a more violent method of breaking cells. Usually, you can use a domestic food processor to break the tissue, and then use an internal knife type tissue masher (that is, a high-speed disperser) of 10000r / min-20000r / min. ) To break up the cells of the tissue, in order to prevent fever and high temperature, it is usually transferred for 10 seconds to 20 seconds, stopped for 10 seconds to 20 seconds, and can be repeated multiple times.
(3) Colloid mill
The basic principle of colloid mill is that the fluid or semi-fluid material is forced to pass between the fixed tooth and the moving tooth at high speed under the action of centrifugal force, so that the material is subjected to strong shear force, friction force, high frequency vibration and high speed vortex, etc. The effect of complex force is effectively crushed, emulsified, homogenized and mixed to achieve a fine and ultra-fine effect. The high-speed relative motion between the fixed rotors is the main condition for the colloid mill to obtain physical fineness. Only by increasing the precision and linear speed of the grinding disc can a good processing effect be achieved.
2. Physical fragmentation
(1) Press
Crushing is a gentle, thorough method of breaking cells. Under a high pressure of 1000 × 105Pa-2000 × 105Pa, tens of milliliters of the cell suspension is suddenly released to atmospheric pressure through a small hole, and the cells will be completely broken. This is an ideal method of breaking cells, but the instrument cost is higher.
(2) Alternating cold and hot
Alternating cold and heat is maintained at 90 ° C for several minutes, immediately placed in an ice bath to cool it, so many times, most cells can be broken, this technology can be used when extracting proteins and nucleic acids from bacteria or viruses.
(3) Repeated freezing and thawing
Repeated freezing and thawing is to cool the cells to be broken to 15 ℃ -20 ℃, and then at room temperature (or 40 ℃) to quickly thaw, so repeated freezing and thawing many times, due to the formation of ice particles in the cells, the salt concentration of the remaining cytosol increases And cause cell disruption.
(4) Ultrasound
Ultrasonic waves use the vibration force of ultrasonic waves to break up cell walls and organelles. When crushing microbial bacteria and yeast, the time is longer, the treatment effect is related to the sample concentration and frequency of use. Pay attention to cooling down during use to prevent overheating.
3. Chemical and biochemical fragmentation
(1) Organic solvent
Organic solvents use fat-soluble solvents such as chloroform, toluene, and acetone, or surfactants such as SDS (sodium dodecyl sulfate) to dissolve the cell membrane, thereby rupturing the cells. This technique can also be used in conjunction with the grinding method.
(2) Autolysis
Autolysis is the storage of fresh biological materials at a certain pH and appropriate temperature. The cell structure dissolves under the action of various hydrolytic enzymes (such as protease and esterase), which releases the contents of the cell. Out, this method is called autolysis. This method should be used with great care, because hydrolytic enzymes can not only destroy cell walls and membranes, but also may decompose certain active ingredients to be extracted.
(3) Swelling
Swelling is in low-osmotic solutions and low-concentration dilute salt solutions. Due to the osmotic pressure difference, a large amount of solvent molecules enter the cell, swelling the cell membrane and releasing the contents of the cell.
(4) Enzymatic hydrolysis
Enzymatic hydrolysis is the use of various hydrolytic enzymes, such as lysozyme, cellulase, snail enzyme and esterase, etc., at 37 ℃, pH8, treatment for 15 minutes, can specifically decompose the cell wall and release the contents of the cell, Enzymatic crushing is suitable for a variety of microorganisms. For example, when extracting plasmid DNA from certain bacterial cells, lysozyme (from egg white) can be used to break the cell wall, while when breaking yeast cells, snail enzyme (from snail) is often used to suspend yeast cells in 0.1mmol / L citric acid Adding 1% snailase to disodium hydrogen phosphate buffer (pH = 5.4) and treating at 30 ℃ for 30 minutes can rupture most of the cell wall. It is better to add 0.2% sparseyl alcohol at the same time. This method can be used in conjunction with grinding
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