In physical disruption methods, the cell membrane is physically broken down by shear or external forces to release cellular components. Although physical methods have traditionally been used to disrupt cells, there are some inherent disadvantages to their use. Localized heating within a sample can occur with many of the techniques described, leading to protein denaturation and aggregation. To avoid this problem, it is essential to pre-chill equipment and keep samples on ice at all times. Reproducibility with homogenization and grinding methods can be challenging due to inexact terminology used to define sample handling. Furthermore, cells disrupt at different times, so the viscosity of the medium constantly changes and released subcellular components are subjected to disruptive forces. In addition to sample handling problems, some physical disruption methods require equipment, such as the French press and sonicator.
Detergent- or solution-based cell lysis is a milder and easier alternative to physical disruption of cell membranes, although it is often used in conjunction with homogenization and mechanical grinding when preparing protein samples from tissues to achieve complete cell disruption. Detergents break the lipid barrier surrounding cells by solubilizing proteins and disrupting lipid-lipid, protein-protein, and protein-lipid interactions. Through empirical testing by trial and error, different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts, and reducing agents have been developed to provide the best possible results for particular species and types of cells. Detergents have both lysing and solubilizing effects.
|Reagent-based methods characteristics||Physical disruption methods characteristics|
|Lysis method||Apparatus||Description||Commonly used with|
|Mechanical||Waring Blender Polytron||Rotating blades grind and disperse cells and tissues||Complex tissues (Liver, muscle)|
|Liquid Homogenization||Dounce Homogenizer Potter-Elvehjem Homogenizer|
|Cell or tissue suspensions are sheared by forcing them through a narrow space|
Dounce & Potter-Elvehjem homogenizers: Small volumes, soft tissues, cultured cells
French press: bacterial cells
|Sonication||Sonicator||High frequency sound waves shear cells||Bacterial or yeast cells|
|Freeze-thaw||Freezer or dry ice with ethanol||Repeated cycles of freezing and thawing disrupt cells through ice crystal formation||Mammalian cells|
|Manual grinding||Mortar and pestle||Grinding tissue, frozen in liquid nitrogen||Hard tissues (e.g., plants), large tissue samples|
Mechanical methods rely on the use of rotating blades to grind and disperse large amounts of complex tissue, such as liver or muscle. The Waring blender and the Polytron are commonly used for this purpose. Unlike the Waring blender, which is similar to a standard household blender, the Polytron draws tissue into a long shaft containing rotating blades. The shafts vary in size to accommodate a wide range of volumes and can be used with samples as small as 1 mL.
Liquid-based homogenization is the most widely used cell disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven PTFE pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. Both homogenizers can be obtained in a variety of sizes to accommodate a range of volumes. A French press consists of a piston that is used to apply high pressure to a sample volume of 40–250 mL, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. The equipment is expensive, but the French press is often the method of choice for breaking bacterial cells mechanically.
|Type||Dounce Homogenizer||Potter-Elvehjem Homogenizer||French Press|
|Benefits||Easy to use with smaller volumes|
Sonication of cells is the third class of physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores, and finely diced tissue. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample. To prevent excessive heating, ultrasonic treatment is applied in multiple short bursts to a sample immersed in an ice bath. Sonication is best suited for volumes <100 mL.
The freeze-thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols.
|Freeze-thaw lysis methods|
Manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant proteins and DNA.
Cells can be treated with various agents to aid the disruption process. Lysis can be promoted by suspending cells in a hypotonic buffer, which cause them to swell and burst more readily under physical shearing. Lysozyme (200 µg/mL) can be used to digest the polysaccharide component of yeast and bacterial cell walls. Alternatively, processing can be expedited by treating cells with glass beads in order to facilitate the crushing of cell walls. This treatment is commonly used with yeast cells. Viscosity of a sample typically increases during lysis due to the release of nucleic acid material. DNase can be added to samples (25–50 µg/mL) along with RNase (50 µg/mL) to reduce this problem. Nuclease treatment is not required for sonicated material since sonication shears chromosomes. Finally, proteolysis can be a problem whenever cells are manipulated; therefore, protease inhibitors should be added to all samples undergoing lysis.
Lysozyme can be used to digest the polysaccharide component of yeast and bacterial cell walls to improve protein extraction efficiency. To demonstrate the benefit of using lysozyme and DNase I to process bacterial extracts, the extractions of two different-sized, over-expressed proteins were compared. Cell pellets from 50 mL culture of E. coli BL-21 over-expressing green fluorescent protein (GFP) or GST-Ral binding protein (GST-RalBP) were lysed using Thermo Scientific B-PER Reagent with and without lysozyme and DNase I. The soluble fractions were separated from the pellets, and the two fractions were analyzed by SDS-PAGE. Although GFP (32 kDa) was extracted equally well in the absence or presence of enzyme, GST-RalBP (75 kDa) required lysozyme and DNase I to be efficiently solubilized.
For Research Use Only. Not for use in diagnostic procedures.