Selected article for: "gel electrophoresis and western blot"
Author: Guo, Huichen; Huang, Mei; Yuan, Quan; Wei, Yanquan; Gao, Yuan; Mao, Lejiao; Gu, Lingjun; Tan, Yong Wah; Zhong, Yanxin; Liu, Dingxiang; Sun, Shiqi
Title: The Important Role of Lipid Raft-Mediated Attachment in the Infection of Cultured Cells by Coronavirus Infectious Bronchitis Virus Beaudette Strain
  • Document date: 2017_1_12
    • ID: 0238bsxb_9
    Snippet: Vero cells treated with or without drugs were incubated with IBV for 24 h at 37ËšC. The cells were scraped in ice-cold PBS and spun down at 20,656 g for 30 s at 4ËšC. The pellet was resuspended in 1 mL of TNEV buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1% Triton X-100) containing a cocktail of protease and phosphatase inhibitors (Roche, Mannheim, Germany). The lysate was kept on ice for 30 min, and then homogenized with 20 stroke.....
    Document: Vero cells treated with or without drugs were incubated with IBV for 24 h at 37˚C. The cells were scraped in ice-cold PBS and spun down at 20,656 g for 30 s at 4˚C. The pellet was resuspended in 1 mL of TNEV buffer (150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1% Triton X-100) containing a cocktail of protease and phosphatase inhibitors (Roche, Mannheim, Germany). The lysate was kept on ice for 30 min, and then homogenized with 20 strokes in a pre-chilled Dounce homogenizer. The supernatant was applied to a discontinuous sucrose gradient followed by centrifugation at 34,426 g for 15 min at 4˚C. Then, the sucrose gradient was centrifuged at 55,000 g for 20 h at 4˚C with SW40Ti rotor (Beckman, USA). Eleven fractions of 1 mL each were collected from top to bottom of the sucrose gradient solution and diluted in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. Then, 10 μL of the samples were subjected to SDS-PAGE and Western blot analysis.

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