Blotting, transfer electrophoresis, is a new method developed in the 1970s. In 1975, Southern established a DNA-RNA hybridization method for detecting specific DNA fragments, which is called Southern blotting. In 1977, Alwine et al applied this method to the research of RNA, called the Southern blotting method. In 1979, Towbin et al. extended the method to protein analysis, called Western blotting. The two-way Western blot method reported by Reinhart in 1982 is called Eastern blotting. At present, Southern, Nouthern, and Western blotting methods are referred to as Southern blotting, Northern blotting, and Western blotting, respectively. Each of the blotting methods can be further divided into a dot blot method and an electrophoretic transfer blot method. The former is to directly adsorb the sample on the solid phase carrier, while the latter is to transfer the sample to the surface of the solid phase carrier after electrophoresis, and the rest of the operation is basically the same. Since macromolecular substances such as nucleic acids and proteins are imprinted into a solid phase carrier, it is easy to chemically or immunologically react with various probes, and thus it is advantageous to detect the physical and chemical properties of certain components in a sample (especially a crude sample). The amount of reagents required for the operation process is small and the sensitivity is high, so the application is relatively common. The basic principle of imprinting (1) concept Application of Blotting (I) Analysis and Preparation of Specific Components General crude products such as macromolecules such as proteins are often subjected to gel electrophoresis and often produce many bands. However, after analysis by the blot transfer procedure, it is possible to find out a specific component that is required, and this special component can be recovered from the corresponding gel segment. The substance obtained by this method has higher purity and the purification step is also simpler. Sampling Cotton Swabs,Nylon Flocked Swab,Small Foam Tip Swab ,Cotton Tipped Swabs Hunan Kangfutai Medical Devices Co., Ltd. , https://www.3kfut.com
1. The probe chemically uses a discriminating substance (such as an antigen, a hormone, a nucleic acid, etc.) and an enzyme (such as horseradish peroxidase) or a nuclides (such as 3H, 35S, 32P), or a fluorescent substance (such as isosulfur The complex formed by the combination of cyanofluorescein, digoxin, etc. is called a probe.
2. A solid phase carrier is a solid material used to adsorb macromolecules of life. Such materials are nitrocellulose (NC) membranes, nylon membranes (NDM), diaxymethylmethyl (DBM) membranes, and diazophenylthiaether (DPT) paper. What is commonly used by zui is an NC film with a pore size of 0.45/lm because of its low cost, strong bonding force and clear background. For the detection of nucleic acids and acidic proteins, the ideal carrier is a positively charged nylon membrane, which has a strong binding force with a negatively charged substance and is simple to handle. However, since it is also easy to combine with negative ion dyes and has a high background value, the use is somewhat limited.
3. Blocking uses a substance that does not react with the analyte (such as protein, nucleic acid, Tween 20, etc.) to block the remaining adsorption sites outside the imprinted region of the carrier, allowing the probe to react with the imprint and not adsorbing onto the carrier. This process is called blocking, so that the background of the imprint is clean and the spots or bands of the imprint are clearer.
4. The blot is transferred by gel electrophoresis or adsorbed to the solid support by direct adsorption or electrophoresis. This process is called electrophoretic blotting, or dot blotting.
5. Autoradiography: The radionuclide-containing complex is placed on X-ray photographic film. When the radionuclide is ionized, a chemical "sensing" effect occurs on the film, which is then developed and fixed to reveal spots or spectra. Belt, this method is more sensitive.
(2) Principle The living macromolecular substance (such as nucleic acid or protein) is imprinted onto the solid phase carrier, and after being treated with the quenching reagent, it can be reacted with the corresponding probe (enzyme label), followed by rinsing with an appropriate solution, and containing the bottom. When the color of the solution is developed, the band can be seen. If the probe used is a radionuclide label, the rinsed carrier should be autoradiographed to detect specific components in the sample.
(B) Detection of interactions between macromolecules of life Different interactions between life macromolecules occur frequently, especially in organisms. Therefore, establishing an effective method for detecting the interaction between macromolecules of life has always been an important topic for people to study. By blotting, interactions between substances such as DNA-RNA, DNA-protein, RNA-protein, enzyme-substrate, glycoprotein-lectin, hormone-receptor can be detected, and this method can also be used to find Corresponding ligands for various macromolecular substances