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Introduction
Cell lysis is the first step in cell fractionation and protein purification and as such opens the door to a myriad of biological studies. Many techniques are available for the disruption of cells, including physical and detergent-based methods. Historically, physical lysis has been the method of choice for cell disruption; however, it often requires expensive, cumbersome equipment and involves protocols that can be difficult to repeat due to variability in the apparatus (such as loose-fitting compared with tight-fitting homogenization pestles). In recent years, detergent-based lysis has become very popular due to ease of use, low cost and efficient protocols. Pierce offers several detergent-based Poppers Reagents for the preparation of whole and fractionated cell lysates that are faster and more convenient than traditional lysis methods.
All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Lipids comprising the plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that associate spontaneously to form a closed bimolecular sheet (Figure 1). Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type.
In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of extracellular barrier confer shape and rigidity to the cells. Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.
Clearly, the technique chosen for the disruption of cells, whether physical or detergent-based, must take into consideration the origin of the cells or tissues being examined and the inherent ease or difficulty in disrupting their outer layer(s). In addition, the method must be compatible with the amount of material to be processed and the intended downstream applications. A summary of both non-detergent and detergent-based lysis techniques follows.
Figure 1.
Lipid bilayer comprising outer plasma membrane of a cell.
Cell Lysis Using Traditional (Non-detergent) Methods
Several methods are commonly used to physically lyse cells, including mechanical disruption, liquid homogenization, high frequency sound waves, freeze/thaw cycles and manual grinding (Table 1). These methods have been reviewed extensively.
Table 1. Techniques used for the physical disruption of cells.
Lysis Method Description Apparatus
Mechanical Waring Blender
Polytron Rotating blades grind and disperse cells and tissues
Liquid Homogenization Dounce Homogenizer
Potter-Elvehjem Homogenizer
French Press Cell or tissue suspensions are sheared by forcing them through a narrow space
Sonication Sonicator High frequency sound waves shear cells
Freeze/Thaw Freezer or dry ice/ethanol Repeated cycles of freezing and thawing disrupt cells through ice crystal formation
Manual grinding Mortar and pestle Grinding plant tissue, frozen in liquid nitrogen
Mechanical Disruption
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 Homogenization
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 Teflon 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 to 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.
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Sonication
Sonication 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.
Freeze/Thaw
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.
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Mortar and Pestle
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.
Additives/Facilitators
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) (Product # 89833, 89834) 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 (Product # 89835) 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. A detailed discussion of protease inhibitors follows later in this section.
Disadvantages of Traditional Lysis Methods
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 fairly expensive equipment, such as the French press and sonicator.
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