Молекулалық биологияда жасушаларды бұзу буфері

Buffer to break cells in molecular biology

A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells (e. g. western blot for protein, or for DNA extraction). Most lysis buffers contain buffering salts (e. g. Tris HCl) and ionic salts (e. g. NaCl) to regulate the pH and osmolarity of the lysate. Sometimes detergents (such as Triton X 100 or SDS) are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells.

The primary purpose of lysis buffer is isolating the molecules of interest and keeping them in a stable environment. For proteins, for some experiments, the target proteins should be completely denatured, while in some other experiments the target protein should remain folded and functional. Different proteins also have different properties and are found in different cellular environments. Thus, it is essential to choose the best buffer based on the purpose and design of the experiments. The important factors to be considered are: pH, ionic strength, usage of detergent, protease inhibitors to prevent proteolytic processes. For example, detergent addition is necessary when lysing Gram negative bacteria, but not for Gram positive bacteria. It is common that a protease inhibitor is added to lysis buffer, along with other enzyme inhibitors of choice, such as a phosphatase inhibitor when studying proteins with phosphorylation.

Buffer creates an environment for isolated proteins. Each buffer choice has a specific pH range, so the buffer should be chosen based on whether the experiment's target protein is stable under a certain pH. Also, for buffers with similar pH ranges, it is important to consider whether the buffer is compatible with the experiment's target protein. The table below contains several most commonly used buffers and their pH ranges. Although detergents are widely used and have similar functions, the physical and chemical properties of detergents of interest must be considered in light of the goals of an experiment. Detergents are often categorized as nonionic, anionic, cationic, or zwitterionic, based on their hydrophilic head group feature. Detergents are a major ingredient that determines the lysis strength of a given lysis buffer.

One common issue faced by many cell lysis buffers is the disruption of protein structures during the lysis process, partially caused by use of detergents. Detergents often prevent the restoration of native conditions necessary for proper protein folding. For the longest time, after a detergent based cell lysis, a buffer exchange and/or dialysis had to be performed to remove the detergent among other hindering compounds to restore native conditions. To overcome this a solution has emerged in the form of a detergent free cell lysis buffer. The GentleLys buffer employs copolymers instead of detergents, ensuring efficient cell lysis while maintaining the native environment crucial for the correct folding of cellular components, such as proteins.

The GentleLys buffer employs synthetic nanodisc copolymers to gently disrupt the cell membrane, offering a milder alternative to conventional detergent based lysis buffers. This gentle approach eliminates the need for harsh chemicals, creating an environment that preserves the native state of cellular proteins. Consequently, the proteins maintain their structural integrity and functionality, a marked departure from the denaturing effects of detergent based buffers.

In studies like DNA fingerprinting the lysis buffer is used for DNA isolation. Dish soap can be used in a pinch to break down the cell and nuclear membranes, allowing the DNA to be released. Other such lysis buffers include the proprietary Qiagen product Buffer P2.