Alkaline protein display assay in cells
Alkaline protein display assay in cells
Different amino acids have side-chain groups with different chemical properties, some with basic side-chains, some with acidic side-chains, which makes different proteins composed of amino acids have different numbers of basic and acidic groups, which will make proteins have different net charges in different pH solutions. If, under physiological conditions, the entire protein molecule is more negatively charged, the protein is acidic; more positively charged, the protein is basic. Therefore, after the nucleic acids are extracted from the specimen by acid treatment, it can be stained with a negatively charged alkaline staining solution, solid green (pH 8.2-8.5), so that the positively charged alkaline proteins will be revealed in this pH environment.
Principle
The basic principle of the alkaline protein display experiment is that the most abundant alkaline proteins in cells are histones, which are tightly wrapped with DNA to form a complex called chromatin, which exists in the nucleus of eukaryotic cells as a storage carrier of genetic information. Histones are synthesized in the cytoplasm during the S phase of the cell cycle, and after synthesis, they are rapidly transported into the nucleus by the nuclear pore to complete the assembly with DNA. Therefore, the basic proteins displayed in the cell are more often found in the nucleus.
Operation method
Alkaline protein display assay in cells
Principle
The basic principle of the alkaline protein display experiment is that the most abundant alkaline proteins in cells are histones, which are tightly wrapped with DNA to form a complex called chromatin, which exists in the nucleus of eukaryotic cells as a storage carrier of genetic information. Histones are synthesized in the cytoplasm during the S phase of the cell cycle, and after synthesis, they are rapidly transported into the nucleus by the nuclear pore to complete the assembly with DNA. Therefore, the basic proteins displayed in the cell are more often found in the nucleus.
Materials and Instruments
Equipment: Move The basic procedure of the alkaline protein display assay in cells can be divided into the following steps: Caveat 1. After the action of trichloroacetic acid carriers must be fully and thoroughly rinsed with running water, so as not to interfere with the solid green staining. For more product details, please visit Aladdin Scientific website.
Dissecting equipment, wax tray, staining cylinder, slides, coverslips, pipettes, absorbent filter paper, ordinary light microscope, thermostatic water bath, toadstool.
Reagents:
① 5% trichloroacetic acid.
② 0.1% basic solid green; ③ 70% ethanol.
③ 70% ethanol.
A. Take 1 toad and execute it by the method of pounding the spinal cord, fix it on a wax disk with its ventral surface facing upwards, cut open the chest cavity and open the pericardium. Cut a small slit in the heart, take 1 drop of blood from the heart and put it on one end of the slide, push the slide, and let it dry at room temperature.
B. Immerse the smear in 70% ethanol for 5 min, and let it dry at room temperature.
C. Immerse it in 5% TCA, and then take it to a water bath at 60 ℃ for 30 min.
D. Rinse it well under running water to remove TCA, and then remove the residual water with a filter paper.
E. Immerse it in 0.1% alkaline solid green staining for 15 min.
F. Rinse it under running water. Dry at room temperature.
G. Microscopic examination or 1 drop of neutral gum, cover the film with a cover slip for sealing and observation.