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