Cell disruption procedures

The recovery of intracellular proteins involves distinct cell disruption procedures, depending on the cell characteristics. For the processing of animal cells, which do not have a cellular wall, mild and moderate techniques are commonly used. Mild techniques include cell lysis by enzymatic digestion, chemical solubilization or autolysis and the use of manual homogenizers and grinders, whereas the moderate techniques involve blade homogenizers and abrasive grinding. 

In general, the physical structure of the tissue must be broken down mechanically followed by an extraction procedure, before the sample can be analyzed. Homogenization using blenders, probe homogenizers, cell disrupters, sonicators, or pestle grinders is particularly useful for muscle, liver, and kidney samples. Regardless of the method used for tissue disruption, the pulse, volume of extraction solvent added, and temperature should be validated and standardized in order to ensure reproducible analytical results. During cell disruption, care should be taken to avoid heat build-up in the sample, because the analyte may be heat labile. 

The interferon is produced within the E. coli cells, which must first be separated from the culture media by using centrifugation. The isolated cells are then solubilized by cell disruption, after which the fraction containing interferon is concentrated by salting-out. Two subsequent procedures using immunoaffinity and cation-exchange chromatography raise the purity of the interferon 1000-fold. 

After cell disruption, gross fractionation of the properly stabilized, crude cell homogenate may be achieved by physical methods, specifically centrifugation. Figure 7.11, Chapter 7, outlines the stepwise procedure commonly used to separate subcellular organelles such as nuclei, mitochondria, lysosomes, and microsomes. 

The only disadvantage of the succinylation procedure (which is practical and amenable to conventional cell disruption processes) is that the final product is a succinylated protein. Succinyl groups cannot be removed from the succinylated proteins under mild conditions. This could be a problem if succinylated yeast protein was a major source of dietary proteins. Therefore we explored the feasibility of using reversible modifying reagents (citraconic anhydride and maleic anhydride) to separate proteins from NAs and subsequently remove the modifying groups under mild acidic conditions. 

To isolate a functional macromolecular component from bacterial cells, you must accomplish three things. First, you must efficiently disrupt the bacterial cell wall and cell-membrane system to facilitate extraction of desired components. Second, you must work under conditions that either inhibit or destroy the many degradative enzymes (nucleases, proteases) released during cell disruption. Finally, you must employ a fractionation procedure that separates the desired macromolecule from other cellular components in satisfactory yield and purity. 

Techniques Careful cell disruption and specific extraction procedures may lower the... 

After recovery of biomass by centrifugation, aldolase-containing cell lysates are obtained by mechanical or ultrasound cell disruption. A purification procedure is usually required to prepare a pure catalyst for synthetic purposes and to avoid the presence of any enzyme producing undesirable side-reactions. Usually, aldolases are purified from cell lysates by a combination of conventional purification steps gel filtration, ammonium sulphate precipitation, ionic exchange chromatography. 

Ultrasonic cell disrupters are manufactured by a half-dozen or so companies. In the author s lab, the Model 550 Sonic Dismenbrator (Fisher Scientific) has been in use. It becomes important to retune the generator when a new probe is changed. There are also additional tuning procedures to follow for microtips. 

Ultrasonication is another liquid-shear method of cell disruption. Ultrasonic vibrations having frequencies greater than 18 kHz are able to disrupt microbial cells in suspension. The ultrasonic vibration could be emitted continuously or in the form of short pulses. A frequency of 25 kHz is commonly used for cell disruption. The duration of this procedure depends on the cell type, the sample size and the cell concentration. The transmission of sonic waves creates a continuous cycle of microbubble cavitation in the suspending medium. These cavities or small bubbles of dissolved gases or... 

The existence of permeability barriers would not be a serious obstacle were it not for the fact that it has proved so far impossible to prepare fully respiring material from disintegrated microorganisms. Although it is possible, by using low temperatures and reinforcing with cofactors, to maintain respiration quantitatively after the disruption of animal cells, these procedures lead to loss of 80% to 90% of respiration when applied to yeast, for example. 

Lehner Mitosis is a risky procedure it involves the disruption of cell—cell contacts, for instance. Broken chromosomes can get lost during mitosis. If it is not worth the effort, why do it ... 

Solute uptake can also be evaluated in isolated cell suspensions, cell mono-layers, and enterocyte membrane vesicles. In these preparations, uptake is normalized by enzyme activity and/or protein concentration. While the isolation of cells in suspension preparations is an experimentally easy procedure, disruption of cell monolayers causes dedifferentiation and mucosal-to-serosal polarity is lost. While cell monolayers from culture have become a popular drug absorption screening tool, differences in drug metabolism and carrier-mediated absorption [70], export, and paracellular transport may be cell-type- and condition-depen-dent. 

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