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Centrifugation
Published in Pau Loke Show, Chien Wei Ooi, Tau Chuan Ling, Bioprocess Engineering, 2019
Differential centrifugation or pelleting is the simplest and most commonly used method for centrifugal separation of biological entities. It is a commonly employed technique used for separating biologically active substances, such as animal and plant viruses, various subcellular fractions (nuclei, chloroplasts, and mitochondria), as well as crude extraction and concentration of biological macromolecules (nucleic acids and proteins) (Graham, 2001a). Differential centrifugation generally uses fixed-angle rotors to separate particles of different sizes and densities by gradually increasing the centrifugal speed. First, the heaviest particles completely sink to the bottom of the tube during low-speed centrifugation. The supernatant is then precipitated by centrifugation at slightly higher rotational speed to obtain a secondary particle sample. By gradually increasing the centrifugal rotation speed, sample particles of different weights can be obtained to achieve the purpose of separation. During each step of operation, light particles close to the bottom of the tube cause some interference and contaminate the precipitate. To avoid this situation, it is necessary to resuspend the precipitate and repeat this centrifugation step several times to obtain particles of uniform size.
Magnetic Nanoparticles for Organelle Separation
Published in Nguyễn T. K. Thanh, Clinical Applications of Magnetic Nanoparticles, 2018
Mari Takahashi, Shinya Maenosono
Isolation of target cellular organelles has attracted attention as a complementary technique because it enables direct analysis of intact proteins on/in the isolated organelles. Several commonly used centrifugation techniques for organelle isolation are briefly described here. To isolate cellular organelles by centrifugation, the cell membrane must be first disrupted by one of the following methods: (i) osmotic shock method, (ii) sonication method, (iii) mild detergent method, (iv) French press method or (v) tissue grinder method (Figure 12.4).14 All of these methods have advantages and disadvantages. For example, physical disruption methods often cause deformation and/or aggregation of proteins owing to heat produced by the equipment and thus need to be performed at low temperature. In addition, these methods are poorly reproducible in terms of cell membrane disruption efficiency. Meanwhile, chemical disruption methods require strict experimental procedures under appropriate conditions; otherwise, the proteins on the cellular organelles can become dissolved. The solution obtained after gentle disruption of the cell membrane, designated the cell homogenate, contains intact cellular organelles. Differential centrifugation is a widely used technique to isolate certain cellular organelles. First, the homogenate is centrifuged at low speed to separate intact cells, nuclei and cytoskeletons from other cellular components. Second, the supernatant is centrifuged at medium speed to precipitate mitochondria, lysosomes and peroxisomes. Third, the supernatant is centrifuged at high speed to obtain microsomes and other small vesicles. Finally, the supernatant is centrifuged at very high speed to obtain ribosomes, viruses and large macromolecules, as shown in Figure 12.5a.
Accumulation, subcellular distribution, and ecological risk assessment of Pb and Cd in Bellamya aeruginosa from the Xiangjiang River, China
Published in Chemistry and Ecology, 2020
Jun Liu, Wei Huang, Zhiliang Li, Jingsong Hu, Yunhua Zhu, Hongyan Xie, Cuiying Peng
Homogenised samples were separated using differential centrifugation at 4°C into five fractions: nuclei, cell membrane, mitochondria, lysosomes, and microsomes. The first fraction was obtained by gentle centrifugation of the tissue homogenate for 10 min at 700×g to remove cell membranes and nuclei. The pellet was used to isolate the cell membranes and nuclei, and the supernatant (S1) was used for the further fractionation of the organelles. Insoluble metal-containing granules were extracted from the nuclei/debris pellet through digestion of the tissue pellet with NaOH followed by centrifugation at 5,000×g to precipitate the granules. The supernatant, S1, was separated by sequential centrifugation into mitochondrial (10 min at 10,000×g), lysosomes (10 min at 16,300×g), and microsomal (30 min at 100,000×g) fractions. Three replicates were analysed in all cases.
Exogenous spermidine elevating cadmium tolerance in Salix matsudana involves cadmium detoxification and antioxidant defense
Published in International Journal of Phytoremediation, 2019
Chunfang Tang, Riqing Zhang, Xinjiang Hu, Jinfeng Song, Bing Li, Danling Ou, Xi Hu, Yunlin Zhao
The extraction of Cd in subcellular fractions of samples adopted the method described in Weigel and Jäger (1980) with some modification. The samples of 1.5 g were broken with liquid nitrogen and homogenized with a 20 mL of pre-cold extraction buffer containing 0.25 mmol/L of sucrose, 50 mmol/L of Tris–HCl with pH value of 4, and 1.0 mmol/L of C4H10O2S2 and MgCl2. The obtained samples were used for the detection of Cd concentration in fresh leaves. The cell wall fraction was the residue obtained after centrifuged at 2500 g and 4 °C for 30 min in a centrifuged tube of 50 mL. The supernatant was further centrifuged at 10,000 g and 4 °C for 45 min using a differential centrifugation (HITACHI CR22GII, Honshu Island, Japan), the resulting remains were the organelle fraction, and the supernatant was the cytosol and vacuole fraction.
Subcellular distribution, chemical forms, and physiological response to cadmium stress in Hydrilla verticillata
Published in International Journal of Phytoremediation, 2019
Guoxin Li, Qingsong Li, Lei Wang, Guoyuan Chen, Dandan Zhang
A total of 0.20 g of fresh plant material was homogenized in pre-chilled extraction buffer (0.25 M sucrose, Tris-HCl buffer solution [pH 7.5], and 1.0 mM DL-dithioerythritol) using a chilled pestle and mortar. The cells were separated into three fractions, namely, the cell wall fraction, the soluble fraction, and the organelle-containing fraction, using a differential centrifugation technique, as suggested by Weigel and Jager (1980), with some modifications. The homogenate was centrifuged at 1250 × g for 15 min. The precipitate was designated as the cell wall fraction and mainly consisted of cell walls and cell wall debris. The resulting supernatant solution was further centrifuged at 15,000 × g for 45 min. The resulting precipitate was referred to as the cell organelle fraction, whereas the supernatant solution was referred to as the soluble fraction. All steps were performed at 4 °C. The three fractions were dried and wet digested for Cd analysis.