Actin is an important cytoskeletal protein of cells, a major protein component in striated muscle fibers, and a major component of muscle filaments and cytoskeletal microfilaments. Actin has a contractile function and is widely distributed. It plays an important role in life activities such as cell secretion, phagocytosis, migration, cytoplasmic circulation and cytoplasmic division. Β-actin (β-Actin) consists of 375 aa and has a molecular weight of about 42 Kd. Its antibodies are mainly used to label smooth muscle and tumors from which it originates. The expression level of β-actin protein in cells is usually not changed, so it is widely used as a reference for consistent sampling in Western countries, and is often used for immunostaining to observe the microfilament structure of cells.
The cytoskeleton is a three-dimensional network structure interwoven with protein fibers. It fills the entire cytoplasmic space and has a structural relationship with the outer membrane and the inner nuclear membrane to maintain cell-specific shapes and cells. Microtubule: It is the main scaffold in the cell and guides the direction of intracellular material transport;
Microfilament: maintains cell morphological characteristics, enables cells to move and contract; is a spiral fiber of 5-8 nm in diameter composed of actin, which is ubiquitous in eukaryotic cells.
Intermediate fiber: It is the cytoskeleton component that plays a major supporting role, giving the cells tension and shear resistance.
Actin is a medium-sized protein consisting of 375-377 amino acid residues and encoded by a large, highly conserved gene. It has a widespread distribution in the biological world. In addition to muscle tissue, it is also found in almost all eukaryotic cells, such as human brain tissue, platelets and various plant cells, which are also found in many fungi.
The primary structure changes of actin in different species are very small. For example, the amino acid sequence of actin in rabbit, bovine and chicken skeletal muscles is only 3-5 amino acids and rabbit rib muscles are different. Both bovine skeletal muscle and myocardium are different. The difference in actin is only the 298th and 375th. These actin genes apparently evolved from the same ancestral gene.
The microfilaments can be assembled. When ATP is bonded to the monomer, there is a high mutual affinity, and the monomer tends to polymerize into a polymer, which is assembly. When ATP is hydrolyzed to ADP, the monomer affinity decreases and the polymer tends to depolymerize, ie, disassemble. The assembly speed of the two ends of the microfilament is not the same, and the positive electrode and the negative electrode are 5-10 times faster. When the ATP concentration reaches a certain critical value, it can be observed that the positive electrode is assembled and the negative electrode is assembled at the same time, and it is called "treading".
1 microfilament is involved in the formation of cytoskeleton; 2 constitutes cell cortex; 3 microfilament is involved in cell movement; 4 microfilament is involved in cell division; 5 microfilament is involved in muscle contraction; 6 is involved in intracellular material transport; 7 microfilament is involved in intracellular signal transduction guide.
Actin and its β-actin antibody
actin is one of the six known isomers of actin, a cytoskeletal protein consisting of 375 amino acids with a molecular weight of about 42 kDa. Widely distributed in the cytoplasm, the expression is very abundant. Actin, a protein encoded by a housekeeping gene, is highly conserved among different species. Therefore, it is often used as an internal reference in most tissues and cells, ie, internal reference.
Actin antibodies recognize only β-actin in WB or immunostaining and cannot be used to detect β-actin in the muscle or skeletal muscle of adult animals.
The protein level of β-actin antibody usually does not change, so it is widely used in WB when the loading amount is consistently referenced. It is also often used for immunostaining to observe the microfilament structure of cells. When used as a reference for Western, the main difference between the Actin antibody and the ubulin antibody is that the molecular weights of the proteins recognized by the two are different, so that a suitable reference can be selected on the same gel on the same membrane, as well as the target protein and the reference protein.
In the literature "Effects of trehalose on the mRNA transcription level and protein expression of β-actin in low temperature storage skin", it was found that the novel cryoprotectant (CPA) trehalose expressed β-actin expression and mRNA transcription level of skin skeleton protein. The effect of regulation is to understand whether the addition of trehalose to a conventional cryoprotectant is superior to that of a conventional cryoprotectant.
Immunohistochemical staining and Western blot were used to observe the expression of β-actin protein. RT-PCR was used to study the β-actin gene level of skin after preservation by different cryoprotectants. Fresh adult skin is divided into 4 groups, which are respectively trehalose-dimethyl sulfoxide (TD), dimethyl sulfoxide-propylene glycol (DP), dimethyl sulfoxide-de-sera keratinocyte culture solution (DK), DMEM. As a cryoprotectant, the liquid nitrogen was stored at -196 °C for 7 days and 14 days, and the fresh skin was used as the control group. The results showed that the expression of β-actin protein and gene transcription in the T-D group were similar to those in the fresh group, and the D-P effect was second. The other two groups were significantly lower than the fresh group. It is concluded that the addition of trehalose to traditional cryoprotectants is superior to traditional cryoprotectants in the protection of β-actin expression in low temperature storage skin.
Many years of research have confirmed that after treatment with traditional cryoprotectants, skin tissue still inevitably has low temperature damage, resulting in a decrease in cell viability. In order to further study the new protective agent for cryopreservation of the skin, the vitality of the skin after preservation is improved. The effect of trehalose on mRNA transcription and protein expression of β-actin in hypothermia was observed. Immunohistochemistry results: β-actin in fresh skin was brown after DAB staining, and the color was evenly distributed. The protein staining was light yellow at 1 week in the DMEM group, and partially negative at 2 weeks, and the cytoplasm was stained blue. Compared with the fresh skin group, although the protein coloration of the T-D group was gradually reduced after two weeks of storage, the morphology of the nucleus was almost normal, the structure was clear, and the color was uniform.
The traditional skin cryopreservation agent mainly increases the viscosity of the whole cell and the concentration of intracellular solute by infiltrating into the cell, reduces the freezing point of the extracellular fluid and the cell contents, and delays the formation speed of the ice crystal. Trehalose is a non-permeable cryoprotective drug discovered and applied in recent years. Toner et al found that the morphological structure of fetal skin tissue stored by using trehalose-dimethyl sulfoxide as a protective agent is stable and effectively improves the vitality of skin storage. This study showed that the skin tissue was treated with trehalose-dimethyl sulfoxide, dimethyl sulfoxide-propylene glycol, dimethyl sulfoxide-KSKF, DMEM, and the expression of β-actin in fresh skin tissue. The highest, the expression of β-actin in the skin after cryopreservation after immersion in trehalose-dimethyl sulfoxide was similar to that of the fresh group, followed by dimethyl sulfoxide-propylene glycol, and the other groups were significantly weakened.
From the related literature reports and experimental results, we analyzed that trehalose showed good protective effect in this group of skin cryopreservation experiments, which may be related to its anti-oxygen free radical action and its excellent vitrification characteristics. After treatment with trehalose, the glass transition temperature increases or approaches -30°C, which is significantly higher than the glass transition temperature of conventional osmotic protective agents. During the freezing process, trehalose tightly encloses the protein structure to form a sugar glass body, which is an amorphous solid state. The formation of such an amorphous structure results in a decrease in molecular motion in the matrix, a decrease in the diffusion coefficient, a slowing in the rate of chemical reaction, and a low relaxation rate in various processes. Especially when the temperature is much lower than the glass transition temperature, the performance is more obvious. The relaxation process of displacement diffusion restriction, such as protein denaturation, is inhibited, and the mobility is lowered, and the structure of protein molecules is tighter, thus resisting the influence of low temperature environment. Comparison of β-actin protein content and gene expression in different types of CPA treatment showed that different CPA interventions had different protective effects on skin tissue, and trehalose combined with dimethyl sulfoxide was superior to other cryoprotectants. The mechanism of protection of trehalose on skin tissue at the genetic level needs further exploration, which provides a more effective scientific basis for trehalose as a skin cryoprotectant.