China
Africa
Explore chapters and articles related to this topic
Biochemistry Of The Murine Ia Antigens
Published in Soldano Ferrone, Chella S. David, Ia Antigens, 2019
Both limited proteolytic digestion and detergent solubilization have been employed in obtaining murine Ia antigens for study. The majority of laboratories studying the biochemistry of murine Ia antigens have employed detergents to solubilize the Ia Antigens. Although both ionic and nonionic detergents will dissolve the lipid bilayer and thus solubilize the Ia Antigens, most workers in the field have chosen to use nonionic detergents. The most frequent choice is Nonidet® P-40 (NP-40) with Triton® X-100 as a second choice. Solubilization of plasma membranes with nonionic detergents produces intact Ia molecules including the membrane-binding portions of the a and (β chains and the associated Ii chain.
Distribution and Biological Functions of Pyruvate Carboxylase in Nature
Published in D. B. Keech, J. C. Wallace, Pyruvate Carboxylase, 2018
Latency of the mitochondrial pyruvate carboxylase activity to its substrates is readily overcome by disruption of the inner mitochondrial membrane by freeze-drying,57 by freezing and thawing,366 by sonication, or with detergents. Both non-ionic detergents (viz., 0.5% NP-40;16 0.06 to 0.5% Triton X-100;518,700,746 Lubrol WX [0.5 to 1 mg/mg protein]173) and deoxycholate (0.1 to 1.0%)212,246,808 have been used successfully, but there is species variation in the optimum concentration to be used.805
Cyclic Nucleotide Metabolism and Action During Senescence
Published in Richard C. Adelman, George S. Roth, Endocrine and Neuroendocrine Mechanisms of Aging, 2017
The most commonly used method of detecting specific protein phosphorylation involves separation of 32P-labeled proteins by Polyacrylamide gel electrophoresis and quantitation by slicing and counting or autoradiography and densitometry. Labeling may be done by incubation of tissue with phosphate-free media containing carrier-free 32Pi, followed by treatment with the appropriate stimulus, or by incubation of homogenates or subcellular fractions with [33Ρ]-γ-ΑΤΡ of high specific activity in the presence and absence of a cyclic nucleotide or Ca2+. In either case, incubations are terminated as quickly as possible, usually by heating with added sodium dodecyl sulfate (SDS).108 Some distinction between particulate and cytosolic proteins can be made by homogenizing labeled tissue with a buffered solution containing sucrose, NaF (50 mM), NaHP04 (10 mM), EDTA (10 mM), and protease inhibitors to preserve states of phosphorylation. A major problem incurred with intact cells exists in the 32P-RNA that is generated and appears as a high background in the gels, thus interfering with measurement of low level phosphorylation of minor proteins. Extraction with the above solution of inhibitors plus NP-40, a detergent which does not readily solubilize nuclear but does solubilize plasma membranes, and removal of nuclei by centrifugation may partially alleviate this problem. Alternatively, treatment of samples with an RNAse that remains active in 0.3% SDS and mercaptoethanol may also help to reduce this back-ground.109
Knockdown of circRNA Paralemmin 2 Ameliorates Lipopolysaccharide-induced Murine Lung Epithelial Cell Injury by Sponging miR-330-5p to Reduce ROCK2 Expression
Published in Immunological Investigations, 2022
Yi Ren, Liang Li, Mengmeng Wang, Zhizhou Yang, Zhaorui Sun, Wei Zhang, Liping Cao, Shinan Nie
All proteins from tissues or cells were extracted using NP-40 lysis buffer (Beyotime, Shanghai, China). The lysates were mixed with loading buffer (Phygene, Fuzhou, China) and denatured at 95°C for 10 min. Subsequently, SDS polyacrylamide gels (Phygene) were used to separate protein bands. The proteins were wet-transferred onto nitrocellulose membranes (Pall Life Science, Beijing, China). Aspecific signals were blocked by 0.5% bovine serum album, and then the proteins were incubated with the primary antibodies, including anti-B-cell lymphoma-2 (anti-Bcl-2; 1:1000; Cusabio Biotech, Wuhan, China), anti-Bax (1:2000; Affinity, Nanjing, China), anti-cleaved caspase 3 (1:1500; Affinity), anti-ROCK2 (1:1000; Cusabio Biotech) and anti-β-actin (1:5000; Affinity). The membranes were incubated with secondary antibodies (1:8000; Affinity), and the protein blots were developed with eyoECL Plus (Beyotime). β-actin was employed for the normalization of protein expression.
LncRNA AC061961.2 overexpression inhibited endoplasmic reticulum stress induced apoptosis in dilated cardiomyopathy rats and cardiomyocytes via activating wnt/β-catenin pathway
Published in Journal of Receptors and Signal Transduction, 2021
Zhibing Qiu, Wen Chen, Yafeng Liu, Ben Jiang, Li Yin, Xin Chen
For the extraction of total protein, it was extracted from hearts tissues and cardiomyocytes. In brief, after we obtained the hearts tissues and transfected cardiomyocytes, NP-40 (P0013F, Beyotime) were used to incubate with the tissues and cells for 15 min at room temperature. After centrifuged for 20 min (14000 × g), the supernatant which was total protein was collected. For the extraction of nucleus protein, it was also extracted from hearts tissues and cardiomyocytes using Nuclear Protein Extraction Kit (P001327F, Beyotime). In brief, after we obtained the hearts tissues and transfected cardiomyocytes, buffer A was mixed with the tissues and cells and vortex for 5 s. Buffer B was also added into the cells and tissues and vortex for 5 s. After centrifuged for 5 min (14000 × g) at 4 °C, collected the supernatant which was nucleus protein.
Lichens exerts an anti-proliferative effect on human breast and lung cancer cells through induction of apoptosis
Published in Drug and Chemical Toxicology, 2021
Sule Ozturk, Merve Erkisa, Seyhan Oran, Engin Ulukaya, Serap Celikler, Ferda Ari
Cytokeratin 18 (CK-18) is cleaved by specific caspases in apoptosis, followed by the occurrence of new CK-18 fragments (M30 antigens) whose concentrations are measured by an immunoassay kit (M30-Apoptosense ELISA kit, Peviva AB, Sweden). 10 000 cells were grown in a 96-well plate in 200 μl culture medium in triplicate and then treated for 72 h with 100 µg/ml of lichen extracts. As a positive control for cell death, paclitaxel (3.12 μM) was used because this agent is considered an appropriate apoptosis-inducer. Cells were exposed to 10% NP-40 for lysis for 10 min on a shaker at the end of the treatment. Wells were centrifuged at 2000 rpm for 10 s to remove the debris, followed by the placement of samples into wells that are coated with a mouse monoclonal antibody as a catcher. After cleansing the plate wells, M30 antibody that is horseradish peroxidase-conjugated was used for detection. An ELISA reader was used to read the absorbances at 450 nm (FLASH Scan S12, Eisfeld, Germany).