Selected article for: "acid protein and lysis buffer"
Author: Li, Lv; Dai, Hong-Juan; Ye, Mao; Wang, Shu-Ling; Xiao, Xiao-Juan; Zheng, Jie; Chen, Hui-Yong; Luo, Yu-hao; Liu, Jing
Title: Lycorine induces cell-cycle arrest in the G0/G1 phase in K562 cells via HDAC inhibition
  • Document date: 2012_11_23
    • ID: 13amcxh2_32
    Snippet: Exponentially growing K562 cells treated with various concentrations of lycorine (1.25, 2.5, or 5.0 μM) or without lycorine were cultivated at 5 × 10 5 cells/mL in several culture flasks (BioCoat). After 24 h of culture, the cells were pelleted by centrifugation, washed three times with PBS, resuspended in 100 μL of RIPA lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.5% deoxycholate, 0.1% SDS, 5 mM NaF, and 0.5.....
    Document: Exponentially growing K562 cells treated with various concentrations of lycorine (1.25, 2.5, or 5.0 μM) or without lycorine were cultivated at 5 × 10 5 cells/mL in several culture flasks (BioCoat). After 24 h of culture, the cells were pelleted by centrifugation, washed three times with PBS, resuspended in 100 μL of RIPA lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl, 1 mM EDTA, 1% Nonidet P40, 0.5% deoxycholate, 0.1% SDS, 5 mM NaF, and 0.5% cocktail), and centrifuged at 13000 rpm and 4°C for 15 min to collect the supernatant. The supernatant protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientific). Equal amounts of protein (50 μg) from each group were electrophoresed for 2 h on 10% sodium dodecyl sulfatepolyacrylamide gels and then transferred to a PVDF membrane (Millipore) using an electroblotter for 100 min at 4°C. Membranes were blocked in PBS with 0.1% Tween 20 (PBST) containing 5% non-fat dried milk power for 1 h. An antibody (Abcam) raised against α-tubulin (1:20000), an antibody (Bioworld) raised against pRB (1:2000) , an antibody (Bioss) raised against p21 an antibody (Cell Signaling) raised against phosphorylated pRB (1:3000), and antibodies (Santa Cruz Biotech) raised against p53 (1:3000), cyclin D1 (1:200), CDK4 (1:200), and CDK2 (1:500) were diluted in PBST containing 5% non-fat milk and membranes were incubated overnight at 4°C. After washing four times with PBST for 10 min each time, the blot was incubated with anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Millipore, 1:5000 dilution in PBST containing 5% non-fat milk) for 1 h at room temperature. After washing three times with PBST for 10 min each time, the blots were developed with a chemiluninescene detection kit (ECL; Millipore), and the optical density of each band was quantified by densitometric scanning.

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