Cell Lysis and Fractionation Support—Getting Started

Historically, physical lysis was the method of choice for cell disruption and extraction of cellular contents; however, it often requires expensive, cumbersome equipment and involves protocols that can be difficult to repeat due to variability in the apparatus (such as loose-fitting compared with tight-fitting homogenization pestles). Also, traditional physical disruption methods are not conducive for high-throughput and smaller volumes typical of modern laboratory research.

In recent years, detergent-based cell lysis methods have become the norm. Through empirical testing by trial and error, different detergent-based solutions composed of particular types and concentrations of detergents, buffers, salts and reducing agents have been developed to provide the best possible results for particular species and types of cells.

Detergents have both lysing and solubilizing effects.

Cell lysis is the first step in cell fractionation, organelle isolation, and protein extraction and purification. As such, cell lysis opens the door to a myriad of proteomics research methods. Many techniques have been developed and used to obtain the best possible yield and purity for different species of organisms, sample types (cells or tissue), and target molecule or subcellular structure. Subcellular fractionation and protein enrichment are important methods in the rapidly growing field of proteomics. Isolation of subcellular fractions and concentration of proteins in low abundance allow for more efficient identification and study of proteins of interest. Examples are the isolation of integral membrane proteins and nuclear proteins.

Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking proteinprotein interaction. These detergents are considered harsh. Non-denaturing detergents can be divided into nonionic detergents (i.e., Triton™ X-100), bile salts (i.e., cholate), and zwitterionic detergents (i.e., CHAPS). These detergents do not denature proteins and do not break protein–protein interactions. These detergents are considered mild.

Detergents are amphipathic molecules, meaning they contain both a nonpolar “tail” having aliphatic or aromatic character and a polar “head.” Like the components of biological membranes, detergents have hydrophobic-associating properties as a result of their nonpolar tail groups. Nevertheless, detergents are themselves water soluble.

Consequently, detergent molecules allow the dispersion (miscibility) of water-insoluble, hydrophobic compounds into aqueous media, including the extraction and solubilization of membrane proteins. Detergent monomers solubilize membrane proteins by partitioning into the membrane bilayer. With increasing amounts of detergents, membranes undergo various stages of solubilization.

Typical cell lysis reagents contain a detergent for protein solubilization and stabilization, a buffer to improve the extraction efficiency and stabilization of the protein, and a salt to improve solubility and stability and to facilitate selective compartment extraction.

Additional components can include reducing agents, chelators, crowding agents, and protease inhibitors.

Isolation of organelles or subcellular fractions allows for segregation, and enrichment of proteins allows for more efficient identification and study of proteins of interest. It also allows for protein localization assessment.

Certain detergents can be used to selectively extract and isolate membrane (hydrophobic) proteins from cytosolic (hydrophilic) proteins. The Mem-PER™ Plus Membrane Protein Extraction Kit (Cat. No. 89842) enriches for integral membrane proteins and membrane-associated proteins from cultured mammalian cells or tissue via selective solubilization using a mild detergent-based protocol. The use of selective detergent extraction eliminates the hassle of phase separation based on hydrophobicity, allowing better reproducibility and higher throughput. The cells are first permeabilized with a mild detergent, allowing the release of soluble cytosolic proteins, after which a second detergent solubilizes membrane proteins.

Several methods, including mechanical disruption, liquid homogenization, sonication, freeze/thaw cycles, and manual grinding are commonly used to physically lyse cells.

• Mechanical disruption: mechanical methods rely on the use of rotating blades to grind and disperse large amounts of complex tissue.
• Liquid homogenization: cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. The number of strokes and the speed at which the strokes are administered influences the effectiveness of homogenization methods.
• Sonication: pulsed, high frequency sound waves agitate and lyse cells, bacteria, spores, and finely diced tissue.
• The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample.
• Freeze/thaw: this technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37˚C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis. This method is commonly used to lyse bacterial and mammalian cells.
• Mortar and pestle: manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle.

Cell lysis disturbs the carefully controlled cellular environment, allowing endogenous proteases and phosphatases to become unregulated. As a result extracted proteins become degraded or artifactually modified by the activities of these molecules.

Please see the various lysis buffers we offer for total protein extraction from mammalian cells.

We offer the B-PER™ Bacterial Protein Extraction Reagent for lysis of bacterial cells.

We offer several formulations for lysis of bacterial cells.

Isolation of subcellular fractions and concentration of low-abundantce proteins allows for more efficient identification and study of the proteins of interest.

We offer the following kits for cell fractionation:

• Mem-PER™ Plus Membrane Protein Extraction Kit, Cat. No. 89842
• NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, Cat. No. 78835
• Subcellular Protein Fractionation Kit for Cultured Cells, Cat. No. 78840
• Subcellular Protein Fractionation Kit for Tissues, Cat. No. 87790
• Mitochondrial Isolation Kit for Cultured Cells, Cat. No. 89874
• Mitochondrial Isolation kit for Tissue, Cat. No. 89801
• Lysosome Enrichment Kit for Tissues and Cultured Cells, Cat. No. 89839

We offer the following kits for cell isolation:

• Primary Cardiomyocyte Isolation Kit, Cat. No. 88281
• Mouse Embryonic Fibroblast Isolation Kit, Cat. No. 88279
• Primary Neuron Isolation Kit, Cat. No. 88280

Detergents are amphipathic molecules containing both a nonpolar “tail” having aliphatic or aromatic character, and a polar “head”. The ionic character of the polar head group forms the basis for broad classification of detergents as ionic, nonionic, or zwitterionic.

Ionic detergents, or those that carry a charge, are the most likely to be denaturing to proteins. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergents totally disrupt membranes and denature proteins by breaking protein–protein interactions through changes in the three-dimensional structure of the proteins. Nondenaturing detergents can be divided into nonionic detergents (i.e., Triton™ X-100), bile salts (i.e., cholate), and zwitterionic detergents (i.e., CHAPS).