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Lysis & Protein Extraction

Cell lysis and extraction for preparation of proteins from cultured mammalian cells, insect cells, Gram positive and Gram negative bacteria, yeast, plant, and other samples.

When purifying proteins for functional or structural studies, the first step is to disrupt the cells or tissue sample and extract the relevant proteins. Cell lysis and protein solubilization are key to effective analysis. The sample may contain thousands of proteins in addition to nucleic acids, polysaccharides, and lipids, all of which can interfere with the analysis. The choice of extraction method depends primarily on the sample and whether the analysis targets all the proteins in a cell or only a component from a particular subcellular fraction.

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Numerous methods are available for disrupting cells and preparing their contents for analysis. In general, gentle methods are employed when the sample consists of easily lysed cultured cells or blood cells, whereas more vigorous methods are employed for the disruption of more robust bacterial or plant cells, or mammalian cells embedded in connective tissue.

  • Detergent-based lysis: Detergent lysis is most frequently used for mammalian cells. Cell suspensions are gently centrifuged and resuspended in lysis solution containing detergent. The membranes are solubilized, lysing cells and liberating their contents.
  • Freeze-thaw lysis: This method is applicable to suspensions of mammalian or bacterial cells. The cell suspension is rapidly frozen using liquid nitrogen. The sample is then thawed and resuspended by pipetting or gentle vortexing in lysis buffer, repeating the process several times. Between cycles the sample is centrifuged, and the supernatant containing soluble protein is retained.
  • Osmotic shock: This is a very gentle method that may be sufficient for the lysis of suspended mammalian or bacterial cells without the use of a detergent. The method, often combined with mechanical disruption, relies on changing from high to low osmotic medium, and is well-suited to applications in which the lysate is to be subsequently fractionated into subcellular components.
  • Ultrasonication: This method of protein extraction is most frequently applied to cell suspensions. Cells are disrupted by high-frequency sound waves via a probe inserted in the sample. The sound waves generate a region of low pressure, causing disruption of the cell membranes.
  • Mechanical methods: Proteins may be extracted from cells and tissues using various crude but effective "crushing and grinding" measures. For example, cell membranes may be disrupted by liquid shear forces using Dounce or Potter-Elvehjem homogenization. Tissues may be homogenized by chopping or mincing in chilled buffer using a Waring blender or Polytron® homogenizer. Tissues or cells may be frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle with alumina or sand. Rapid agitation of cells with fine glass beads disrupts cell walls, effective for most Gram positive and Gram negative bacteria.
  • Enzymatic digestion: Enzymatic methods are frequently used when extracting proteins from bacteria, yeast, or eukaryotic cells embedded in fibrous tissues where cell membranes are surrounded by a robust protective structure. Cell lysis enzymes dissolve cell walls, coats, capsules, capsids, or other structures not easily sheared by mechanical methods alone. Enzymatic digestion is often followed by homogenization, sonication, or vigorous vortexing in a lysis buffer.

Additionally, because endogenous proteases and phosphatases may be liberated upon cell disruption and degrade the target molecule, the sample should be protected during cell disruption and subsequent purification using protease and phosphatase inhibitors to avoid uncontrolled loss of target.