Ideally, cell lysis and protein solubilization are carried out in the electrophoresis sample buffer. Interactions involved in protein aggregation must be broken for successful electrophoresis. Alternative detergents can be employed to target different subcellular components (see below). Subcellular fractionation can be achieved by differential centrifugation and in some instances, by using specific lysis detergents.Īs an example, proteins that are known to be hydrophobic (membrane bound) can be separated from hydrophilic proteins (cytoplasmic) by incubation of cells with non-ionic detergents (Triton-X or Tween 20) which results in the formation of two distinct layers a hydrophobic and a hydrophilic. In such cases, one can employ cellular fractionation to isolate specific cell organelles to achieve more optimal detection on western blot. Often times, the protein of interest is present at low levels or present in tissues that contain many other proteins making detection difficult. See Our Protein Extraction Products » Cell Fractionation Best suited for soft, solid tissuesĪpplication of gentle abrasion by vortexing cells with glass beadsĬell Lysis Volume Recommendations Type of cellsĪdd 2 ml and sonicate or dounce homogenizeĪdd 10 volumes, then sonicate or vortex with glass beads Mortar usually filled with liquid nitrogen during grinding to keep sample cold Grinding of sample to fine powder with mortar and pestle. Cool on ice to avoid overheatingĪpplication of shear forces by forcing a cell suspension through a small orifice at high pressure Usually followed by another disruption method, such as sonicationĭisruption of cell suspension by short bursts of ultrasonic waves. Suspension of cells in iso-osmotic solutions containing enzymes that digest cell wall of plants, yeast, or bacteria. Suspension of cells in hypotonic solution cells swell and burst, releasing cellular contentsįreezing in liquid nitrogen and subsequent thawing of cells Techniques for Specific Sample Types Suitability of cell disruption methods to various sample types. Centrifuge extracts extensively (20,000 x g for 15 minutes at 15☌). Regardless of cell disruption method, remove any insoluble material to avoid blocking the pores of the electrophoresis gel. Enzyme treatment is usually followed by another disruption method such as sonication. For example, cellulase and pectinase for plant cells, lyticase for yeast cells, and lysozyme for bacterial cells. Often both chemical disruption and some sort of mechanical method are used together to maximize protein yields.Įnzymes that digest cell walls of plants or bacteria aid in cell lysis. For grinding with mortar and pestle, addition of splashes of liquid nitrogen will keep the sample cool. During sonication, immersion in an ice bath is sufficient. Some methods like sonication generate heat which can overheat the sample so use cooling to avoid overheating. Involve exposing cells to physical forces using dounces, sonicators, or grinding of tissue. Zwitterionic detergents like CHAPS and ionic detergents like SDS are more effective at solubilization and denature proteins during lysis. Non-ionic detergents like NP-40 are mild and nondenaturing but do not solubilize hydrophobic proteins well. Simple exposure to detergents can lyse cells that disrupt easily, like blood cells or tissue culture cells. Lastly, the appropriate choice of lysis buffer can yield higher amounts of your target protein as the detergents in the buffer can affect lysis efficiency and solubilization of different proteins.Second, consider the subcellular location of your protein of interest and choose a method that enriches for that cellular compartment.First, consider the method of cell disruption as different sample types require different methods for efficient cell lysis.For effective cell lysis that yields your protein of interest in a soluble form, there are a few factors to consider:
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