The precise components and procedure required for making a cell lysis solution depends on several factors, including the type of cells and the objective of the experiment. This BiologyWise article explains how to make cell lysis solution with respect to the major ingredients, and how they vary with different experimental setups.
Sodium dodecyl sulfate (SDS), the most commonly used detergent for cell lysis and protein denaturation, is also widely used in several soaps, shampoos, laundry detergents, and toothpastes.
A cell lysis solution is a detergent-based buffer solution used to break open the desired cells and further isolate a particular cellular component of interest. It is also referred to as a cell lysis buffer or simply, lysis buffer. This process of lysing cells using chemical agents is termed as chemical disruption.
Apart from detergent and buffering agents, additional agents are added that aid the lysing process, eliminate unwanted cellular components, and/or protect the desired cellular component. These additions depend on the cell type involved, the cellular component to be studied, and the precise techniques that need to be performed on the lysate.
Given below is the basic procedure to prepare a cell lysis solution, followed by a description of the components required to prepare the solutions, and their variations with respect to the commonly followed protocols for different experiments.
Procedure to Make a Cell Lysis Solution
Given below is the procedure to prepare a lysis solution containing 10mM Tris-HCl buffer, 1mM EDTA as the chelating agent, and 0.5% SDS as the detergent.
|Step 1: Preparation of 1 L of 1 M Tris-HCl (pH 8) stock solution
Dissolve 121 g Tris-HCL (molecular weight = 157.60 g) in 800 ml distilled water, adjust the pH to 8 using HCl solution, and make up the volume to 1 L using distilled water.
Step 2: Preparation of 500 ml of 0.5 M EDTA stock solution
Dissolve 93.0 g of EDTA [EDTA.Na2.2H2O] (molecular weight = 372.24 g)] in 400 ml of distilled water, add 10 g (approx.) NaOH pellet to adjust pH to 8, and make up the volume to 500 ml using distilled water.
Step 3: Preparation of 10% SDS stock solution
Dissolve 10 g of SDS in 90 ml distilled water, and make up the volume to 100 ml using distilled water.
Step 4: Preparation of 500 ml of the Tris-EDTA SDS lysis buffer
Add 5 ml of 1 M Tris-HCl (pH 8), 1 ml 0.5 M EDTA, and 5 ml of 10% SDS solution to 400 ml of distilled water. Make up the volume to 500 ml.
Choosing the Reagents for a Cell Lysis Buffer
All cell lysis solutions are prepared using a suitable buffer solution, so as to maintain the appropriate pH. Disruption of the cells will disturb the internal pH of the cell, which alters the structural integrity of proteins and other macromolecules. In order to avoid this, generally a buffer solution adjusted at a pH equal to the normal intracellular pH is used.
A buffer solution comprises a weak base and its conjugate acid, or a weak acid and its conjugate base. Each buffering agent can work as an effective buffer only within a specific pH range called the buffer range. The choice of buffering agent depends on this buffer range. Generally, the buffers are used at a concentration of 10 – 50 mM and are adjusted to pH 7.4 – 8.
The two commonly used buffers for making cell lysis solutions, and their buffer range are as follows:
Cell membranes are made up of phospholipid layers which are held together through polar interactions. A detergent serves as an emulsifier and disrupts these polar interactions. It also forms complexes with the lipid molecules and protein molecules embedded in the membrane, and precipitates them out of the solution.
Detergents can be grouped as ionic or non-ionic, depending on the nature of the hydrophilic group, or as mild or strong depending on their ability to solubilize membranes and proteins. Ionic detergents with positively charged groups are termed cationic detergents, and those with negatively charged groups are termed anionic detergents.
Mild detergents are used when cells need to be lysed for purifying and/or studying cellular proteins, so as to avoid denaturation of the desired proteins. On the other hand, strong detergents that denature cellular proteins are used while lysing cells for DNA isolation.
Given below are some of the commonly used detergents, their application as cell lysing agents, and usual concentrations in which they are used for cell lysis.
Role: The chelating agent is a chemical that sequesters divalent cations, like Mg++, and Ca++, which are required for membrane stability. Due to such chelation, these cations are no longer associated with the membrane, thereby, weakening it. Moreover, the lysing of membrane results in exposing the nucleic acids to nucleases, which are otherwise sequestered inside the lysosomes. These nucleases are inactive in the absence of divalent ions. Thus, chelating agents protect the DNA and RNA molecules from degradation by these nucleases.
Types and Choice: EDTA (Ethylenediaminetetraacetic acid) and EGTA (ethylene glycol tetraacetic acid) are the widely used chelating agents. The latter one has a lower affinity for Mg++ ions, as compared to EDTA. Hence, it is used when Ca++ ions need to be selectively sequestered.
Osmotic Stabilizers: Glucose, sucrose, sorbitol, and glycerol may be added for osmotic stability, and preventing the cellular components from osmotic shock that may occur during the sudden rupturing of cell membrane. In addition, they also help stabilize lysosomal membranes, thereby, reducing the release of degradative enzymes from the lysosomal lumen.
Salts: Disruption of cells and the release of cellular components into the medium, may alter the ionic strength of the medium. In order to deal with this and maintain ionic strength, salts, like sodium chloride [NaCl], potassium chloride [KCl], and ammonium sulfate [(NH4)2SO4] may be added.
Enzymes: Depending on the target macromolecules to be isolated from cells, lytic enzymes may be added to either protect them from the action of other enzymes, or degrade the remaining macromolecules that may contaminate the lysate.
For example, in case of protein purification, nucleases may be added to degrade the nucleic acids of the cell. Also, protease inhibitors may be added to prevent the breakdown of proteins. On the other hand, in case of isolation of nucleic acids, proteases may be added to degrade the proteins and eliminate them. In case of DNA isolation, RNases may be added to prevent contamination by RNA molecules. While dealing with bacterial cells, lysozyme is added to lyse cell walls by cleaving the peptidoglycan linkages present in their cell walls.
Cell lysis solutions are used for the chemical disruption of cells, and may also be used in a combination with other methods, like homogenization, sonication, grinding, freeze-thaw techniques, etc. This is especially useful for lysis of fungal cells and plant cells, since they have strong cell walls around the cell membranes.