A cell lysis buffer is a critical first component to any isolation protocol. It is fundamental to the first step of protein or nucleic acid extraction as it aids in the chemical breakdown of cell membranes and compartments, enabling target molecules to leave the cell. There are many types of lysis buffers; most are easy to make, but most are also commercially available. They are often included in kits for immunoprecipitation, co-ip protocol, nucleic acid isolation, and others. When using a lysis buffer for protein capture the addition of protease inhibitors is generally recommended in order to protect proteins.
The cell lysis buffer must disrupt membrane chemistry but preserve target molecules
The type of lysis buffer used is dictated by the goal of the experiment. However, most cell lysis buffers will contain a detergent that disrupts the lipid bilayer of the cell membrane. If the experimental goal requires preservation of tertiary protein structure, such as a co-ip, then the cell lysis buffer needs to be non-denaturing. Non-denaturing buffers typically contain a non-ionic detergent like NP-40 or Triton X-100. These are surfactants, with the chemical composition of soap, that disrupt the fatty acid bilayer. These non-denaturing buffers will have low salt concentrations, usually less than 120 mM NaCl. However, some salt is needed to prevent non-specific protein interactions. Ethylenediaminetetraacetic acid, or EDTA, is a common additive that has multiple functions including protease inhibition and protection against oxidative damage. Tris is another additive used to buffer the pH and prevent protein denaturation.
Detergent free lysis buffers are available for lysis protocols including some type of mechanical disruption of the cell or tissue. Common techniques for mechanical disruption include sonication and vortexing.
Sometimes a denaturing buffer is required to extract proteins. If this is the case then sodium dodecyl sulfate (SDS) and sodium-deoxycholate detergents are included in the cell lysis buffer. Denaturing detergents will sometimes include heating to 95ºC
General procedure for performing cell lysis
- First determine the cell lysis buffer composition that best suits the goals of the experiment. If the cells are in a culture dish, add the lysis buffer and scrape off the cells.
- There will be an optimal incubation time for the buffer to work. If isolating molecules from whole tissue, then a mechanical lysis step may be needed to aid in membrane breakdown.
- After incubation of the cell or tissue with the lysis buffer, the sample is spun down in a centrifuge. The supernatant is collected while the pellet is discarded.
- The cell lysate can then be used immediately for the remainder of the experimental protocol or frozen and stored for later use.
What is the purpose of a cell lysis buffer
The word lysis comes from the greek word for “loosen.” Cell lysis is the process of rupturing the membrane or walls of a cell. The purpose of a cell lysis buffer is to use a chemical mixture to disrupt the exterior environment of a cell in a way that causes it to break open and release its contents.
How do I choose a lysis buffer and make a cell lysis buffer
The lysis buffer you need depends on what materials you are interested in extracting from the cell. There are several components to consider for your cell lysis buffer such as pH, salts, detergents, whether or not to use protease inhibitors, To make the cell lysis buffer you will want to choose a buffer that is compatible with your protein, usually Tris-HCl or HEPES-NaOH, which have different pH ranges that also need to be considered. You can also use salts such as NaCl or KCl to increase the ionic strength of your buffer, which will disrupt molecular interactions. Detergents come in varying strengths and are chosen based on whether extracted proteins need to be denatured or not. Sodium dodecyl sulfate is an ionic detergent that is strong enough to solubilize proteins and denature them. A weaker detergent such as NP-40 can be used to solubilize proteins but not denature them. Lastly, a protease inhibitor is included when there is danger of proteases cleaving your protein of interest.
Using Magnetic Separation for a Cell Lysis Buffer
Magnetic separation can be used for (1) capture the DNA or intracellular proteins after the lysis or (2) for separate the cells from the raw suspension. In this second case is very important to keep the cells intact, then the lysis can be performed in a clean buffer, then removing interferences. Using constant magnetic force magnetic separation systems (as Sepmag) improves both the capture efficiency and the integrity of the cells .