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Cell fragmentation technology guide
Cell fragmentation technology refers to the use of external force to destroy the cell membrane and cell wall, so that the cell contents including the target product components released technology, is the basis for the separation and purification of non-secretory biochemical substances (products) synthesized by cell contents. Combined with significant advances in recombinant DNA technology and tissue culture techniques, proteins previously thought to be difficult to obtain can now be mass-produced.
A variety of cell breaking methods have been developed to accommodate different uses and different types of cell wall breaking. The crushing method can be classified into two categories: mechanical method and non-mechanical method. Let's learn it together.
Mechanical method:
1. High pressure homogenization
A high pressure homogenizer is a commonly used device that consists of a positive displacenemt pump that generates high pressure and a discharge valve that has narrow holes and can be adjusted in size. The cell slurry enters the pump body through the check valve, forcing it to rush out at high speed in the small hole of the discharge valve under high pressure, and shoots to the impact ring, due to sudden decompression and high-speed impact, so that the cell is broken by high liquid phase shear force. In the mode of operation, it can be used single through the homogenizer or multiple cycles, and it can also be continuously operated. In order to control the rise of temperature, the temperature can be adjusted with dry ice at the inlet, so that the outlet temperature is adjusted at about 20 ° C. In industrial-scale cell breakage, the operation method of multiple cycles is often used for cells that are difficult to break and have high concentration or are in the growth rest period, such as yeast.
2. Skaking Bead method
Small tissue samples of equal volume and high density ZircoBeads were put into sealable 2ml screw cap microtubes, then buffer and stabilizing ingredients were added to 1.5ml volume, and then the 6500RPM oscillator was used to vibrate up and down at high speed for 8 seconds, rest for 8 seconds, and vibrate again for 8 seconds. This method is the fastest and can process the most samples at one time. One machine can process up to 2400 samples a day. It is convenient for a small number and variety of people.
3. High speed bead grinding (fine grinding)
Grinding is a commonly used method in which a cell suspension is quickly stirred with an abrasive agent such as glass beads, quartz sand or alumina to break the cells. In industrial-scale crushing, high-speed bead mills are often used.
4. ultrasonication
The cell suspensions were treated with an ultrasonic probe of 15-25 KHZ, which was emitted by an ultrasonic oscillator. There are different types of ultrasonic oscillator, commonly used for the electroacoustic type, it is composed of a generator and a transducer, the generator can produce high-frequency current, the role of the transducer is to convert the electromagnetic oscillation into mechanical vibration. Ultrasonic oscillator can be divided into two types of groove and probe directly inserted into the medium, the general crushing effect of the latter is better than the former.
Non-mechanical method:
1. osmotic shock (osmotic shock)
Osmotic shock is a milder crushing method. The cells are placed in a solution of high osmotic pressure (such as a certain concentration of glycerol or sucrose solution). Due to the osmotic pressure, the water in the cells will seep out, and the cells will contract. The water outside the cell quickly infiltrates into the cell, causing the cell to expand rapidly and rupture.
2. freezing and thawing
The cells are frozen at a low temperature (about -15 ° C) and then melted at room temperature, repeated many times to achieve the wall-breaking effect. Due to freezing, on the one hand, the hydrophobic bond structure of the cell membrane can be broken, thereby increasing the hydrophilicity of the cell, on the other hand, the water in the cell crystallizes, forming ice grains, causing the cell to expand and break. This method can be used for cells with weak cell walls.
3. enzyme lysis
Enzymatic hydrolysis is the use of cell wall dissolving enzymes to treat bacterial cells, so that the cell wall is partially or completely destroyed, then the osmotic pressure impact and other methods to destroy the membrane, further increase the permeability of intracellular products. lysozyme is suitable for the breakdown of gram-negative bacteria cells, and when applied to Gram-positive bacteria, it needs to be supplemented with EDTA to make it act more effectively on the cell wall. The cell wall of eukaryotic cells is different from that of prokaryotic cells and requires different enzymes.
4. chemical treatment
Chemical treatment can dissolve cells or extract intracellular components. Chemical reagents such as acids, bases, surfactants and organic solvents are commonly used
Hengyuan Brand Cell recommendation:
Human embryo skin fibroblasts; CCC-ESF-1
Human microglia; HMC3
Human osteosarcoma cells with luciferase 143B/LUC
Human renal tubular epithelial cells; HKC
Mouse pancreatic cancer cells
Mouse alveolar epithelial cell MLE-12
Mouse small brain cell C8-D1A
Hamster kidney fibroblasts; BHK-21
Chicken embryo fibroblast; UMNSAH/DF-1
Duck embryo fibroblast; Duckembryo
Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory, equipped with professional technical research and development team, long-term commitment to a variety of biological reagents, cells, serum, ELISA kit research and development and sales, experiment to provide free testing services, and provide superb technical services, if you have experimental problems welcome to come to consult, to provide you with professional one-to-one technical guidance.
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