Cell Disruption

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Overview

Cell disruption is the first step for releasing desired biomolecules from within the cell. This section provides details of different cell disruption methods and their suitability for various cell types and also provides some troubleshooting tips.

Related Sections: Protein Solubilization, and Removal of Interfering Substances.

General Considerations

The effectiveness of a cell disruption method determines the accessibility of intracellular proteins for extraction and solubilization. Different biological materials require different cell disruption strategies, which can be divided into two main categories: gentle and harsher methods (see Table below).

  • Use gentle cell disruption protocols when the sample consists of cells that lyse easily, such as red blood cells and tissue culture cells
  • Use harsher methods, which are based mainly on mechanical rupture (see Goldberg 2008 for a review of cell disruption techniques), with biological materials that have tough cell walls (for example, plant cells and tissues and some microbes)
  • When working with a new sample, use at least two different cell disruption protocols and compare their efficiency concerning yield (by protein assay) and qualitative protein content (by SDS-PAGE)
  • Optimize the power settings of mechanical rupture systems and incubation times for all lysis approaches
  • Mechanical cell lysis usually generates heat; use cooling where required to avoid overheating the sample

All of these cell disruption methods cause the release of compartmentalized hydrolases (phosphatases, glycosidases, and proteases) that can alter the protein composition of the lysates. In experiments where relative amounts of protein are to be analyzed, or in experiments involving downstream immunodetection, the data are only meaningful when the protein composition is preserved.

Avoid enzymatic degradation by using one or a combination of the following techniques:

  • Disrupt the sample or place freshly disrupted samples in solutions containing strong denaturing agents such as 7–9 M urea, 2 M thiourea, or 2% SDS. In this environment, enzymatic activity is often negligible
  • Perform cell disruption at low temperatures to diminish enzymatic activity
  • Lyse samples at pH >9 using either sodium carbonate or Tris as a buffering agent in the lysis solution (proteases are often least active at basic pH)
  • Add a chemical protease inhibitor to the lysis buffer. Examples include phenylmethylsulfonylfluoride (PMSF), aminoethylbenzylsulfonylfluoride (AEBSF), tosyllysinechloromethylketone (TLCK), tosylphenylchloromethylketone (TPCK), ethylenediaminetetraacetic acid (EDTA), benzamidine, and peptide protease inhibitors (for example, leupeptin, pepstatin, aprotinin, bestatin). For best results, use a combination of inhibitors in a protease inhibitor cocktail
  • If protein phosphorylation is to be studied, include phosphatase inhibitors such as okadaic acid, calyculin A, and vanadate

Following cell disruption:

  • Check the efficacy of cell wall disruption by light microscopy
  • Centrifuge all extracts extensively (20,000 x g for 15 min at 15°C) to remove any insoluble material

Suitability of Cell Disruption Methods to Various Sample Types

Technique Description Bacteria Yeast,
Algae,
Fungi
Seeds Green
Plant
Material
Soft
Tissues
Mammalian
Cell
Culture
Gentle Methods
Osmotic lysis Suspension of cells in hypotonic solution; cells swell and burst, releasing cellular contents
Freeze-thaw lysis Freezing in liquid nitrogen and subsequent thawing of cells
Detergent lysis Suspension of cells in detergent-containing solution to solubilize the cell membrane; this method is usually followed by another disruption method, such as sonication
Enzymatic lysis Suspension of cells in iso-osmotic solutions containing enzymes that digest the cell wall (for example, cellulase and pectinase for plant cells, lyticase for yeast cells, and lysozyme for bacterial cells); this method is usually followed by another disruption method, such as sonication
Harsher Methods
Sonication Disruption of a cell suspension, cooled on ice to avoid heating and subjected to short bursts of ultrasonic waves
French press Application of shear forces by forcing a cell suspension through a small orifice at high pressure
Grinding Breaking cells of solid tissues and microorganisms with a mortar and pestle; usually, the mortar is filled with liquid nitrogen and the tissue or cells are ground to a fine powder
Mechanical
homogenization
Mechanical homogenization with either a handheld device (for example, Dounce and Potter-Elvehjem homogenizers), blenders, or other motorized devices; this approach is best suited for soft, solid tissues
Glass-bead
homogenization
Application of gentle abrasion by vortexing cells with glass beads

Products for Cell Lysis and Disruption

Bio-Rad's solution to successful and reproducible sample preparation is its MicroRotofor™ lysis kits, which provide cell lysis and protein extraction protocols that are tailored to the specific needs of different sample sources. Bio-Rad offers kits designed for removal of salts, high abundance proteins, and other contaminants. They incorporate procedures such as affinity and size exclusion chromatography to improve resolution of 2-D gels.

MicroRotofor cell lysis kits.

All four kits are based on the same chaotropic protein solubilization buffer (PSB), which contains non-detergent sulfobetaine 201 (NDSB 201) along with urea, thiourea, and CHAPS for particularly effective solubilization. The kits generate total protein samples that are ready to be applied to SDS-PAGE, IEF, and 2-D gel electrophoresis. Different sample types have different requirements for effective cell disruption, and all four kits combine PSB with other elements to accommodate these specific needs.

Troubleshooting

Problem Cause Solution
Laemmli sample buffer turns yellow Sample buffer is too acidic Add Tris base until buffer turns
blue again
Sample very viscous High DNA or carbohydrate content

Fragment DNA with ultrasonic waves during cell lysis and protein solubilization

Add endonucleases like Benzonase

Precipitate protein with TCA/acetone (ReadyPrep™ 2-D cleanup kit) to diminish carbohydrate content

References

Goldberg S (2008). Mechanical/physical methods of cell disruption and tissue homogenization. Methods Mol Biol 424, 3–22.

Related Content

Literature
Number Description Download
3096 Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B Click to download
6194 Protein Sample Generation General Tips Click to download
6195 Protein Sample Preparation (Human Tissue) Click to download
6196 Protein Sample Preparation (Mammalian Tissue) Click to download
6197 Protein Sample Preparation (Plant Leaves) Click to download
6198 Protein Sample Preparation (Microbial Cultures) Click to download
6199 Buffer Formulations Click to download
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