Cellular fractionation of the wild‐type H. pylori cells was performed as described earlier 14 (link). Briefly, bacterial cell extract was centrifuged at 148 000 g in SW‐55 rotor (Beckman coulter ultracentrifuge, Irving, TX, USA) at 4 °C for 1 h, the supernatant was recovered as cytoplasmic/periplasmic fraction (C/P) and the resulting pellet was recovered as total membrane (TM). Volume of C/P was measured and TM was resuspended in the same volume of PBS. Equal volume of each sample was mixed with 2X‐SDS sample buffer, separated in SDS/PAGE, and subjected to western blotting using an appropriate antibody.
Coulter ultracentrifuge
Manufactured by Beckman Coulter
Sourced in United States
The Coulter ultracentrifuge is a high-speed centrifuge designed for the separation and analysis of particles, cells, and macromolecules. It utilizes a rotor that can reach speeds up to 100,000 revolutions per minute, generating high centrifugal forces necessary for the separation of complex biological samples.
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Lab products found in correlation
4 protocols using coulter ultracentrifuge
1
Cellular Fractionation of H. pylori
2
Lipid Raft Isolation and Detection
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Cells were treated with 5 ng/mL IL-1β or PBS for 30 min and lysed in cell lysis buffer (CST) containing 1 mM PMSF. An identical amount of protein from each sample was mixed with 90% sucrose in MES buffer (6 mL final volume, sucrose concentration 51.7–58.7%) and transferred to a Beckman ultracentrifuge tube. Four milliliters of 35% sucrose followed by 3 mL of 5% sucrose were overlaid, the samples were spun in a Beckman Coulter ultracentrifuge (39,000 rpm; approximately 180,000 × g in a SW40Ti rotor, 20 h), and 26 fractions were collected from the top of the gradient. For the detection of the lipid raft fractions, all fractions were dot-blotted with HRP-labeled cholera toxin B (Sigma-Aldrich) to detect Ganglioside GM1.
3
Cell Fractionation and Membrane Analysis
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Hp cells were grown on BHI agar plates, collected, washed twice with PBS and re-suspended in 500 μl of 20 mM Tris-HCl, pH 8.0. Cell fractionation was performed as described earlier [41 (link)]. Briefly, re-suspended cells were sonicated, unbroken cells and debris were removed by centrifugation at 8000 X g for 10 min at 4°C. The supernatant was centrifuged at 148,000 X g for 1 hr at 4°C in a SW-55 rotor, Beckman coulter ultracentrifuge. The supernatant was a mixture of cytoplasmic/periplasmic fractions (C/P), and the pellet was considered to be the total membrane fraction (TM). Fractionated samples were dissolved in 2X SDS sample buffer, boiled and subjected to SDS-PAGE, followed by Western blotting using appropriate antibodies.
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Hp cells were grown on BHI agar plates, collected, washed twice with PBS and re-suspended in 500 μl of 20 mM Tris-HCl, pH 8.0. Cell fractionation was performed as described earlier [41 (link)]. Briefly, re-suspended cells were sonicated, unbroken cells and debris were removed by centrifugation at 8000 X g for 10 min at 4°C. The supernatant was centrifuged at 148,000 X g for 1 hr at 4°C in a SW-55 rotor, Beckman coulter ultracentrifuge. The supernatant was a mixture of cytoplasmic/periplasmic fractions (C/P), and the pellet was considered to be the total membrane fraction (TM). Fractionated samples were dissolved in 2X SDS sample buffer, boiled and subjected to SDS-PAGE, followed by Western blotting using appropriate antibodies.
4
Density Gradient Isolation of Gas6
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