Overview of Cell Lysis and Protein Extraction
Structure and diversity of cells
All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating cell contents from the extracellular environment. Lipids comprising the plasma membrane are amphipathic, having hydrophilic and hydrophobic moieties that associate spontaneously to form a closed bimolecular sheet. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains spanning the hydrophobic core. In addition, peripheral proteins bind the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid head groups. The nature of the lipid and protein content varies with cell type and species of organism.
In animal cells, the plasma membrane is the only barrier separating cell contents from the environment, but in plants and bacteria the plasma membrane is also surrounded by a rigid cell wall. Bacterial cell walls are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble to alkaline conditions. Both of these are surrounded by an outer glycoprotein layer rich in the carbohydrate mannan. Plant cell walls consist of multiple layers of cellulose. These types of extracellular barrier confer shape and rigidity to the cells. Plant cell walls are particularly strong, making them very difficult to disrupt mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption using glass beads, whereas bacterial cell walls are the easiest to break compared to these other cell types. The lack of an extracellular wall in animal cells makes them relatively easy to lyse.
This 50-page handbook provides protocols and technical and product information to help maximize results for protein/gene expression studies. The handbook provides background, helpful hints and troubleshooting advice for cell lysis, protein purification, cell fractionation, protease inhibitors and protein refolding. The handbook is an essential resource for any laboratory studying protein/gene expression.
Cell lysis and protein extraction
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 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.
By careful optimization of physical disruption techniques, detergent-buffer solutions and density gradient methods, procedures have been developed to enable separation of subcellular structures or classes of compounds. For example, with the appropriate detergents, hydrophobic membrane proteins can be solubilized and separated from hydrophilic proteins. A combination of tools and steps enable intact nuclei, mitochondria and other organelles to be isolated for study or protein solubilization.
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.
To prevent these effects and obtain the best possible protein yield in cell lysis, protease and phosphatase inhibitors are added to the lysis reagents. Numerous compounds have been identified and used to inactivate or block the activities of proteases and phosphatases by reversibly or irreversibly binding to them.
When the goal of cell lysis is to purify or test the function of a particular protein, special attention must be given to the effects of the lysis reagents on the stability and function of the protein(s) of interest. Certain detergents will inactivate the function of particular enzymes, and long-term stability of extracted/purified proteins often requires that they be removed from the initial lysis reagents and/or stabilized by addition of particular compounds.
Some cell lysis and protein solubilization methods cause the denaturation of proteins. Functional tests with such proteins requires that they be renatured. Many proteins spontaneously refold into their native, functional structures when the denaturing solubilization reagents are removed by dialysis. Other proteins, however, will fold into non-functional and even insoluble forms by this process. In such cases, specialized sets of buffer conditions must be tested to identify those that promote the highest possible yield of properly refolded protein.
For Research Use Only. Not for use in diagnostic procedures.