Cell lysis is an essential step in the extraction and purification of intracellular proteins, and the method that you use to disrupt your cell samples can determine the success and reproducibility of your downstream applications to a great extent. Keep in mind that one vital component of the starting biological material is inevitably lost in every step of the experiment, so it’s important to get everything you need right from the start. This underscores the need of choosing the cell disruption method that will allow you to extract the best possible yield and purity of your biological materials.
But since there are two major groups of cell disruption protocols (physical or mechanical and chemical cell lysis), how do you know which one to choose? To shed more light on the topic and help you make a more informed decision on the matter, here are some things you need to consider.
Physical or Mechanical Cell Lysis
Physical or mechanical methods of cell disruption have traditionally been the method of choice for cell lysis for two good reasons:
(1) It can be used with a wide variety of materials
(2) The amount of force applied and the duration of the treatment can easily be adjusted to suit the source material.
This method includes grinding, mechanical disruption, freeze-thaw cycles, liquid homogenization, and sonication.
Along with the advantages of this method comes numerous disadvantages, which include the following:
- Some techniques necessitate the need for highly expensive equipment (e.g., French press, sonicator).
- Increased risk for sample handling problems. Since cells disrupt at different times, the released subcellular components are repeatedly subjected to disruptive forces.
- The reproducibility may vary since there is no standard terminology used in defining the handling of samples.
- Increased risk of protein denaturation and aggregation due to localized heating. However, this can be avoided and/or minimized by pre-chilling the equipment and keeping the samples on ice at all times.
- Some of the techniques are complicated and cumbersome to use.
- May not be compatible when working with small volumes and high-throughput.
Mechanical homogenization is ideal for lysing soft, solid tissues. A French press is usually the method of choice for lysing bacterial cells while the mortar and pestle method is the most commonly used method for disrupting plant cells. Sonication works best for lysing cells as well as bacteria, spores and finely diced tissues while the freeze-thaw method is usually used for mammalian and bacterial cells.
In some cases, cells are treated with various chemical additives to facilitate successful physical or mechanical disruption. This is done by suspending the cells in hypotonic buffer causing them to swell and burst more readily when subjected to physical shearing. DNase and RNase can also be added to reduce the viscosity of the sample resulting from the release of nucleic acid during lysis while lysozyme can also be added to aid in the digestion of bacterial and yeast cell walls.
Chemical Cell Lysis
Chemical or enzymatic cell lysis can be achieved by (1) using specific chemicals to disrupt the lipid membrane or cell wall and force the cell to release its contents or (2) suspending the sample in hypotonic solution (e.g., dilute sucrose solution). This is significantly gentler than the mechanical method and is suitable for lysing bacterial, fungal and yeast samples.
Some of the most commonly used chemicals for cell lysis include organic solvents (e.g., alcohols, ether, and chloroform), chelating agents (EDTA), detergents or surfactants (e.g., SDS, Triton) and chaotropic agents (e.g., urea, guanidine).
Advantages of Chemical Cell Lysis
- A gentler and more rapid method for lysing cells.
- Produces more reproducible results.
- Leads to higher protein yields.
- Can be used for various sample types.
Disadvantages of Chemical Cell Lysis
- The cost of using large volumes of reagents needed for large-scale production can be prohibitive.
- When used incorrectly, chemicals and detergents can damage and/or destroy the cell contents.
- The presence of salts and detergents may not be compatible with protein assays. It may also affect the results of downstream applications (e.g., mass spectrometry).
- It may pose significant health and safety risks to the user.
