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