Homogenizer cell disruption

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. 

Teflon pestle glass homogenizer (potter) with rotor (1000—1500 rpm) for efficient and homogeneous cell disruption. 

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. 

Physical Methods Physical methods include mechanical disruption by milling, homogenization, or ultrasonication. Typical high-speed bead mills are composed of a grinding chamber filled with glass or steel beads which are agitated with disks or impellers mounted on a motor-driven shaft. The efficiency of cell disruption in a bead mill depends on the concentration of the cells, the amount and size of beads, and the type and rotation speed of the agitator. The optimum wet solid content for the cell suspension for a bead mill is typically somewhere between 30 percent to 60 percent by volume. The amount of beads in the chamber is 70 percent to 90 percent by... 

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. 

Method of cell disruption E. coli cells are suspended in the binding buffer (0.25 gram cells per mL), and the suspension is passed twice through a homogenizer (Manton Gaulin) at 9000 psi. 

Method of cell disruption cell suspension is passed three times through a high-pressure homogenizer 10,500-13,500 psig. 

Method of cell disruption cell suspension passed three times through high-pressure homogenizer at 10,000 psig Adsorbent STREAMLINE SP in 50 mM sodium acetate pH 5.0 Capturing conditions dry cell concentration, 1.4% suspended in 50 mM sodium acetate pH 5.0, containing endonuclease (benzonase) at a ratio of 10 fiL per 60 g cells (dry weight). 

Intracellular Products. Intracellular production of bioproducts is less preferable but sometimes the only way to produce certain compounds in appreciable amounts. In this case, cell disruption is required for recovery. High-pressure homogenization, bead mills, and chemical or enzymatic disruption of the cell wall with lysozyme or similar enzymes can be used to achieve cell breakage. In the case of small molecules, organic solvent extraction has also been described. If cell debris remains in the centrate, it must be removed by methods described above, thus adding extra steps to the process. 

Figure 5.5 Some methods for the separation and isolation of homogeneous cell populations from tissue fragments. The tissue, composed of three cell types (O, , A), is disrupted and a solution of single cells obtained. The cell types are shown being separated by (/l) buoyant density centrifugation, (0) selective cellular lysis, (C) affinity chromatography, and (D) magnetoseparation.

The composition of the lysis solution is dictated by the nature of the proteins under study and the subsequent techniques applied to the sample. One of the major choices to be made is whether or not a detergent is required at this stage. If the membrane and soluble fractions are to be separated the initial cell disruption protocol should not include a detergent, as many of the membrane proteins would be solubilized. In this case physical disruption of the cells should be used (e.g., sonication of cells or homogenization of tissues). The choice of lysis conditions is a vital consideration in this work, as proteins need to be solubilized while preservation of posttranslational modifications, inhibition of proteases, maintenance of protein-protein interactions, and, if an immunoaffinity purification step is to be performed, suitability for the antibody to function are essential. For example, SDS is very good at solubilizing membrane proteins but... 

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