Description
Cell lysis is the first step in cell fractionation, organelle isolation, and protein extraction and purification. As such, cell lysis and extraction open the door to a myriad of proteomic 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 tissues), and target molecules or subcellular structures.
Structure and diversity of cells.
All cells have a plasma membrane, a protein-lipid bilayer that forms a barrier separating the cellular contents from the extracellular environment. The lipids that make up the plasma membrane are amphipathic and have hydrophilic and hydrophobic moieties that spontaneously associate to form a closed bimolecular sheet. Membrane proteins are embedded in the lipid bilayer, held in place by one or more domains that span the hydrophobic core.
In addition, peripheral proteins are attached to the inner or outer surface of the bilayer through interactions with integral membrane proteins or with polar lipid headgroups. The nature of the lipid and protein content varies depending on the type of cell and the species of organism.
In animal cells, the plasma membrane is the only barrier separating the cell contents from the environment, but in plants and bacteria, the plasma membrane is also surrounded by a rigid cell wall. The walls of bacterial cells are composed of peptidoglycan. Yeast cell walls are composed of two layers of ß-glucan, the inner layer being insoluble under alkaline conditions.
Both are surrounded by an outer layer of glycoprotein rich in mannan carbohydrates. Plant cell walls consist of multiple layers of cellulose. This type of extracellular barrier confers shape and rigidity in cells. Plant cell walls are particularly strong, making them very difficult to break mechanically or chemically. Until recently, efficient lysis of yeast cells required mechanical disruption with glass beads, while 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. There is no universal protocol for protein sample preparation. Sample preparation protocols must take into account several factors, such as sample origin or sample type, chemical and structural heterogeneity of proteins, the cellular or subcellular location of the protein of interest, required protein yield ( which depends on the applications), and the proposed subsequent applications.
For example, body fluids such as urine or plasma are already more or less homogeneous protein solutions with low enzyme activity, and only minor manipulation is required to obtain protein from these samples. However, tissue samples require extensive manipulation to break tissue architecture, control enzyme activity, and solubilize proteins.
The quality or physical form of the isolated protein is also an important consideration when extracting proteins for certain downstream applications. For example, applications such as functional enzyme-linked immunosorbent assay (ELISA) or crystallography require not only intact proteins, but also proteins that are functionally active or retain their 3D structure.
_1672221993__32211.original.jpg "399")
