Technical details of the cell disruption procedure largely depend on the type of tissue or cells to be homogenised. In the case of multicellular organisms, the first aim is to disintegrate the tissue into individual cells by abolishing the connections that organise the cells into the given tissue. Then the plasma membrane and, in case of plants, fungi and bacteria, the cell wall need to be ruptured. The harshness of the treatment can greatly vary depending on the tissue and cell type. For example, in the case of blood cells that do not need to be disintegrated from a solid tissue, even a mild osmotic shock using a hypotonic solution can lead to the rupture of the cell membrane. In the case of cells having a cell wall or tissues stabilised by a strong extracellular matr
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this chapter we survey how individual proteins can be isolated in homogeneous form from biological samples—especially from tissues of multicellular organisms. Naturally, the more information we have on the protein to be investigated, the more straightforward it is to establish a well-suited and efficient isolation protocol.
The first question to consider is the distribution of the given protein among the various tissues of the organism. Obviously, the tissue in which the given protein is the most abundant should be used as the starting point of the isolation protocol. The next question is whether the protein is intracellular, extracellular (secreted) or membrane-bound.
If it is intracellular, the subcellular distribution of the protein should be considered. If the protein is associated to one of the many organelles of the eukaryotic cell, that organelle should be first isolated. The process through which individual organelles (plasma membrane, nucleus, mitochondria, etc.) are separated from one another is called cell fractionation. The process of cell fractionation starts with the disruption of the tissue and its cell constituents by a homogenisation procedure performed as gently as possible. Once the cells are opened up, individual organelle types can be separated from each other by various types of centrifugation techniques.
5.1. Cell disruption
The technical details of the cell disruption procedure largely depend on the type of tissue or cells to be homogenised. In the case of multicellular organisms, the first aim is to disintegrate the tissue into individual cells by abolishing the connections that organise the cells into the given tissue. Then the plasma membrane and, in case of plants, fungi and bacteria, the cell wall need to be ruptured. The harshness of the treatment can greatly vary depending on the tissue and cell type. For example, in the case of blood cells that do not need to be disintegrated from a solid tissue, even a mild osmotic shock using a hypotonic solution can lead to the rupture of the cell membrane. In the case of cells having a cell wall or tissues stabilised by a strong extracellular matrix, simple osmosis-based treatments are inefficient. In such cases various mechanical methods applying shearing force on the cells can be used. The two most frequently applied tools are high-speed laboratory blenders and ultrasonic cell disruptors. While blenders can disrupt even highly structured strong tissues, ultrasonic cell disruptors applying ultrasound (~ 20–50 kHz) to the sample (sonication) are used mostly in the case of cell suspensions. The ultrasonic cell disruptor generates the high-frequency waves electronically. These shock waves are transmitted to the cell suspension via an oscillating metal probe. The oscillation causes large localised pressure inhomogeneity resulting in cavitation eventually disrupting the cells.
There are several other procedures that also use shearing force to open up cells. Some of these apply high pressure to pump the cell suspension through a very narrow channel or orifice into a low pressure container. Due to the sudden drop of pressure the cells “explode” in the container. Shearing force can be also generated by a pair consisting of a carefully designed glass tube and a tightly fitting glass pestle, called the Potter-Elvehjem homogeniser, or a potter in short. The diameter of the tube is just a little larger than that of the pestle. The sample is pushed into the very narrow space between the sides of the tube and the pestle. The shearing force is generated as the cell suspension squeezes up and past the pestle. This method is applied on the suspension of individual cells (already dissociated tissues, blood cells etc.).
Plant cells protected by cell wall are most often disrupted by various grinding methods. Manual grinding is the most common method. The tissue is usually frozen in liquid nitrogen and then crushed using a mortar and pestle.
Optimal cell disruption methods open up a high percentage of the cells in the sample while preserving the organelles or molecules to be investigated in their native state. This is not a trivial task. In order to preserve the native state of most organelles and molecules, the procedure should be quick and the heat generated by the disruption method should be dissipated by intensive cooling. This helps to avoid heat denaturation of proteins and also lowers the rate of unwanted chemical reactions such as oxidation or proteolytic cleavage of proteins. To further suppress these chemical reactions, oxidation can be prevented by the addition of reducing agents and proteolysis can be controlled by the addition of a mixture of protease inhibitors, often referred to as a protease inhibitor cocktail. In order to extract the content of the cell into a native-like solution, the buffers used for cell disruption often mimic the cytosol in terms of pH and ionic strength.
The first question to consider is the distribution of the given protein among the various tissues of the organism. Obviously, the tissue in which the given protein is the most abundant should be used as the starting point of the isolation protocol. The next question is whether the protein is intracellular, extracellular (secreted) or membrane-bound.
If it is intracellular, the subcellular distribution of the protein should be considered. If the protein is associated to one of the many organelles of the eukaryotic cell, that organelle should be first isolated. The process through which individual organelles (plasma membrane, nucleus, mitochondria, etc.) are separated from one another is called cell fractionation. The process of cell fractionation starts with the disruption of the tissue and its cell constituents by a homogenisation procedure performed as gently as possible. Once the cells are opened up, individual organelle types can be separated from each other by various types of centrifugation techniques.
5.1. Cell disruption
The technical details of the cell disruption procedure largely depend on the type of tissue or cells to be homogenised. In the case of multicellular organisms, the first aim is to disintegrate the tissue into individual cells by abolishing the connections that organise the cells into the given tissue. Then the plasma membrane and, in case of plants, fungi and bacteria, the cell wall need to be ruptured. The harshness of the treatment can greatly vary depending on the tissue and cell type. For example, in the case of blood cells that do not need to be disintegrated from a solid tissue, even a mild osmotic shock using a hypotonic solution can lead to the rupture of the cell membrane. In the case of cells having a cell wall or tissues stabilised by a strong extracellular matrix, simple osmosis-based treatments are inefficient. In such cases various mechanical methods applying shearing force on the cells can be used. The two most frequently applied tools are high-speed laboratory blenders and ultrasonic cell disruptors. While blenders can disrupt even highly structured strong tissues, ultrasonic cell disruptors applying ultrasound (~ 20–50 kHz) to the sample (sonication) are used mostly in the case of cell suspensions. The ultrasonic cell disruptor generates the high-frequency waves electronically. These shock waves are transmitted to the cell suspension via an oscillating metal probe. The oscillation causes large localised pressure inhomogeneity resulting in cavitation eventually disrupting the cells.
There are several other procedures that also use shearing force to open up cells. Some of these apply high pressure to pump the cell suspension through a very narrow channel or orifice into a low pressure container. Due to the sudden drop of pressure the cells “explode” in the container. Shearing force can be also generated by a pair consisting of a carefully designed glass tube and a tightly fitting glass pestle, called the Potter-Elvehjem homogeniser, or a potter in short. The diameter of the tube is just a little larger than that of the pestle. The sample is pushed into the very narrow space between the sides of the tube and the pestle. The shearing force is generated as the cell suspension squeezes up and past the pestle. This method is applied on the suspension of individual cells (already dissociated tissues, blood cells etc.).
Plant cells protected by cell wall are most often disrupted by various grinding methods. Manual grinding is the most common method. The tissue is usually frozen in liquid nitrogen and then crushed using a mortar and pestle.
Optimal cell disruption methods open up a high percentage of the cells in the sample while preserving the organelles or molecules to be investigated in their native state. This is not a trivial task. In order to preserve the native state of most organelles and molecules, the procedure should be quick and the heat generated by the disruption method should be dissipated by intensive cooling. This helps to avoid heat denaturation of proteins and also lowers the rate of unwanted chemical reactions such as oxidation or proteolytic cleavage of proteins. To further suppress these chemical reactions, oxidation can be prevented by the addition of reducing agents and proteolysis can be controlled by the addition of a mixture of protease inhibitors, often referred to as a protease inhibitor cocktail. In order to extract the content of the cell into a native-like solution, the buffers used for cell disruption often mimic the cytosol in terms of pH and ionic strength.
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