Ok so it’s science time! Yes, this one is under the Education category of When Women Inspire for good reason. It’s back to biology class for us. Let’s begin with
defining cell lysis. It’s the process of breaking the cell membrane of cells or bacteria down to produce the output of intracellular material, caused by lysines.
What are Cells Made Of?
All cells have a membrane made of phospholipids that separate the cellular content of the extracellular environment. Phospholipids are amphipathic and have membrane proteins embedded in them. The nature of lipids and proteins varies, depending on the cell type.
In the animal cell, the membrane is the only barrier. But in plants and bacteria, the membrane is surrounded by a cell wall. The bacterial cell wall contains peptidoglycans that add shape and stiffness to cells.
About Cell Rupture
Some cells are more difficult to rupture than other kinds. Why rupture or perform cell lysate? For various research and manufacturing reasons. The method must be gentle enough that no damage occurs to the protein. For the purpose of this post’s discussion, the goal is to remove the protein. Also, the method must be compatible with the amount of material to process and the applications of this. Navigate here for tissue lysates.
There is also cytolysis mediated by lymphocytes. Say again? They are cells in the bloodstream that act as defenses. They show on their surface lymphocyte receptors (TCR = T-Cell receptor) that bind to the antigen-molecule complex of the major histocompatibility complex I. As a result, they secrete lymphokines and perforins, which produce ion channels in the infected or neoplastic cell. It then leads to the lysis and subsequent death of the cell. Important to note is that TCRs of the T lymphocytes will recognize the antigens in host cells infected by a virus or in cells that that have progressed from normal to tumor cells.
Types of Lysis
There are two types of lysis: (1) traditional lysis, and (2) lysis by means of detergents. Here’s a link to a good reference.
Traditional methods of cell lysis include:
• Homogenizing liquid. The cells are broken by forcing them to pass through very small spaces.
• Sonification. High frequency waves break cells.
• Freeze. Continuous freezing cycles break the cell inducing the formation of crystals.
As for lysis by way of detergents, it is a milder way to break down the cell membrane. The detergents break the lipid barrier by solubilizing the proteins and interrupting the interaction lipid-lipid, lipid-protein and protein-protein. Detergents, like lipids, associate with each other and bind to hydrophobic surfaces. They consist of a hydrophilic polar head and a non-polar hydrophobic tail.
There is no standard protocol available. The detergent will depend on the application you want to give it. Many studies in which electrophoresis is used typically use SDS to denature the proteins completely. The choice of detergent for cell lysis will also depend on the type of cell. It’s important to also consider the used buffer, the pH, the salt concentration, and the temperature for the choice of the correct detergent.
Material, and Preliminary Steps
At the start of the cell lysate protein extraction process, the material usually consists of biological tissues or cell cultures (eukaryotic or prokaryotic). It can also be inert particles, a mixture in solution, etc.
A lysis step consists of breaking the cell walls and biological membranes, using mechanical actions (crushing, French press, sonication), solubilizing agents (detergents) and other extraction aids, such as protease inhibitors, to protect the proteins of interest.
A solubilization step consists of making the proteins soluble, generally in the aqueous phase. This is typically done by detergents. Examples of detergents are SDS, Triton X-100 or Nonidet, or chaotropic agents to disrupt the protein. This usually occurs in conjunction with lysis.
Some proteins take a long time to dissolve in the detergent. Fractionation steps then follow, using different methods.
The sample then gets mixed with a polar solvent and another apolar, such as an organic solvent, phenol, or chloroform. The proteins get distributed among the different liquid phases according to their respective solubility in each solvent. The lower and/or higher phase is recovered after decantation, a fancy word for separation of the mixture.
Fractionation by Differential Precipitation
One or more proteins of interest, or – just the opposite – undesirable, are precipitated in the presence of certain conditions. Thus, euglobulins or sphase soluble proteins precipitate in aqueous solution in the presence of salts of a specific concentration. For example, IgG immunoglobulins precipitate in the presence of 30-60% ammonium sulfate. Temperature and pH are factors to consider too.
Purification by Depletion and Other Methods
This step involves eliminating undesirable compounds, by methods such as precipitation, adsorption, or affinity. Purification by preparative chromatography is used to separate and purify the proteins according to the retention mechanisms. It must follow one of the following:
- ion exchange that uses charges to separate substances;
- gel filtration chromatography;
- hydrophobic interaction chromatography;
- affinity chromatography
In ion exchange chromatography, the proteins separate based on charges. According to their ionic charge, the proteins will unfix, retain either strong or weakly, and then separate. This is often done in columns with a buffer flow that separates the proteins. Fractions collect over time. Affinity purification, also known as affinity chromatography, focuses on the interaction between the protein and nucleic acid. Macromolecules recognize the acid and selected ones add to its surface to separate them from the others.
As for purification by preparative electrophoresis, the common ways to separate and purify proteins are:
• capillary electrophoresis;
• agarose gel electrophoresis;
• polyacrylamide gel electrophoresis;
• polyacrylamide gel electrophoresis, with sodium dodecyl sulfate;
• Isoelectric focusing
Now you know more about what is cell lysis, and steps involved in protein extraction!