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Is there Tris in the cell lysate?
The presence of cell lysate is often found in the nucleic acid detection material virus transport medium, because the cell lysate plays a vital role in the nucleic acid extraction process. Generally, nucleic acid extraction includes two major steps: lysis and purification of the sample. Lysis is the process of freeing the nucleic acid in the sample in the lysis system, and purification is the complete separation of nucleic acid from other components in the lysis system, such as proteins, salts and other impurities. the process of.
Is there Tris in the cell lysate? The answer is yes. Tris is abbreviated as Tris and it is abbreviated as THAM in foreign countries. Tris is an organic base. Tris buffer should be added to the cell lysate to ensure stable pH during cell lysis. Almost all lysates contain detergents (such as SDS, Triton X-100, NP-40, Tween 20 , etc.) and salts (such as Tris, EDTA, NaCl , etc.). In addition to providing a suitable lysis environment (such as Tris), the role of salt also includes inhibiting the damage of nucleic acid by nuclease in the sample during the lysis process (such as EDTA) and maintaining the stability of nucleic acid structure (such as NaCl) , etc. Detergents are used to denature the protein, destroy the membrane structure and untie the protein connected to the nucleic acid, so that the nucleic acid is released in the lysis system.
Desheng Tris packaging diagram
Most of the active ingredients in the cell lysate are detergents, so what is a detergent? The key to detergent function is to have an amphiphilic structure. All detergents have the characteristics of hydrophilic "head" area and hydrophobic "tail" area. The structure of commonly used anionic detergent SDS is shown in the figure:
These structural properties allow detergents to accumulate in aqueous media. At a sufficiently high concentration, the polar hydrophilic region of each molecule faces the polar solute (water), while the hydrophobic regions gather together to form a thermodynamically stable micelle with a hydrophobic core. The hydrophobic core region of the detergent micelle combines with the hydrophobic surface of the protein to form a soluble protein-detergent complex.
Detergents are classified according to their hydrophilic groups: anionic/cationic detergents, non-ionic amphoteric detergents, and amphoteric detergents.
1. Anionic/cationic detergent: It has a strong effect and will change the structure of protein to a greater extent, and is more susceptible to factors such as pH, ionic strength and the nature of counter ions;
2. Non-ionic detergents: the effect is mild, because they do not separate protein-protein bonds, non-ionic detergents allow proteins to retain their natural structure and function, and are not easily deformed (detergents with shorter hydrophobic chain lengths are easier Cause protein inactivation);
3. Amphoteric detergents: Compared with ionic detergents, it produces less denaturation. Compared with non-ionic detergents, it can more effectively destroy protein-protein bonds and reduce aggregation.
Common types of lysate:
1. NP40 lysis buffer:
(1) The main components are 50mM Tris (pH7.4), 150mM NaCl, 1% NP-40.
(2) Overview: NP-40 is a very mild non-ionic detergent, 1% concentration can basically destroy the cell membrane, but the damage to the nuclear membrane is weak, combined with a specific buffer to obtain cytoplasmic protein . It has strong binding force with protein, and is used to prevent the hydrophobic interaction between material molecules and ensure the full dissolution and structural stability of the protein.
(3) Uses: conventional Western, IP, co-IP and ELISA.
2. SDS lysis buffer:
(1) Main ingredients: 50mM Tris (pH8.1), 1% SDS.
(2) Overview: It is a relatively strong cell tissue lysis solution, which can lyse the nuclear membrane. In SDS-PAGE electrophoresis, SDS can open the hydrogen and hydrophobic bonds of the protein and bind to the protein molecule, so that various protein-SDS complexes are charged with the same density and far exceed the original charge of the protein molecule, thus covering the protein molecules. The difference in charge makes the electrophoretic mobility only depend on the molecular size.
(3) Uses: used for conventional Western, ChIP, etc.