Polysome profiles were performed as described before (Kuzuoğlu-Öztürk et al., 2016 (link)). HEK293T cells were pretreated with cycloheximide (50 μg/ml) for 30 min. Lysates were prepared in lysis buffer (10 mM Tris-HCl pH = 7.4, 10 mM NaCl, 1.5 mM MgCl2, 0.5% Triton X-100, 2 mM DTT, 50 μg/ml cycloheximide) and polysomes separated on a 10%–50% sucrose gradient in gradient buffer (10 mM Tris-HCl pH 7.4, 75 mM KCl, 1.5 mM MgCl2). Polysome fractions were collected using the Teledyne Isco Density Gradient Fractionation System. Protein from sucrose fractions was isolated by methanol extraction. In detail, 4× volumes of MetOH were mixed with the sucrose fractions, then mixed with 1× volume of chloroform and then with 3× volumes of water. After centrifugation, the upper phase was removed leaving the lower and inter-phases which were precipitated using 3× volumes of MetOH. Samples were spun down and the dried pellet dissolved in 2× protein sample buffer. Fractions were analyzed by western blotting.
Isco density gradient fractionation system
Manufactured by Teledyne
The Isco Density Gradient Fractionation System is a laboratory equipment designed for the separation and analysis of macromolecules and particles based on their density. It utilizes a density gradient to facilitate the separation process, allowing for the isolation of specific components within a complex sample.
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Lab products found in correlation
3 protocols using isco density gradient fractionation system
1
Polysome Profiling of HEK293T Cells
2
Polysome Profiling in HEK293T Cells
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Polysome profiles were performed as described before (Kuzuo glu-O ¨zt€ urk et al., 2016) . HEK293T cells were pretreated with cycloheximide (50 mg/ml) for 30 min. Lysates were prepared in lysis buffer (10 mM Tris-HCl pH = 7.4, 10 mM NaCl, 1.5 mM MgCl 2 , 0.5% Triton X-100, 2 mM DTT, 50 mg/ml cycloheximide) and polysomes separated on a 10%-50% sucrose gradient in gradient buffer (10 mM Tris-HCl pH 7.4, 75 mM KCl, 1.5 mM MgCl 2 ). Polysome fractions were collected using the Teledyne Isco Density Gradient Fractionation System. Protein from sucrose fractions was isolated by methanol extraction. In detail, 4x volumes of MetOH were mixed with the sucrose fractions, then mixed with 1x volume of chloroform and then with 3x volumes of water. After centrifugation, the upper phase was removed leaving the lower and inter-phases which were precipitated using 3x volumes of MetOH. Samples were spun down and the dried pellet dissolved in 2x protein sample buffer. Fractions were analyzed by western blotting.
3
Polysome Profiling for Transcriptome Analysis
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Polysome profiles were performed as described before (Kuzuoglu-Ozturk et al., 2016) . HEK293T cells were pre-treated with cycloheximide (50 µg/ml) for 30 min. Lysates were prepared in lysis buffer (10 mM Tris-HCl pH=7.4, 10 mM NaCl, 1.5 mM MgCl 2 , 0.5% Triton X-100, 2 mM DTT, 50 µg/ml cycloheximide) and polysomes separated on a 10-50% sucrose gradient in gradient buffer (10 mM Tris-HCl pH 7.4, 75 mM KCl, 1.5 mM MgCl 2 ).
Polysome fractions were collected using the Teledyne Isco Density Gradient Fractionation System.
To isolate RNA from sucrose fractions, samples were first digested with proteinase K (Sigma Aldrich, 1% of the sample volume; 100 mg/ml in 50 mM Tris-HCl pH=8.1, 10 mM CaCl 2 buffer) at 37°C for 45 min and shaking at 400 rpm. The digested samples were mixed with 1 volume of Phenol:Chloroform:Isoamyl alcohol (PanReac AppliChem, 25:24:1, v/v), vortexed and spun down 5 min at 20,000 g at 4°C. Supernatants were transferred into 3 volumes of 100% ethanol, 0.1 volumes of 3M NaOAc pH=5.2 and 1 µl of GlycoBlue, and precipitated at -20°C. Samples were pelleted for 30 min at 20,000 g and 4°C, washed once with 100% ethanol and another time with 70% ethanol, dried and resuspended in 30 µl H 2 O.
Fractions were reverse transcribed and analysed by RT-qPCR.
Polysome fractions were collected using the Teledyne Isco Density Gradient Fractionation System.
To isolate RNA from sucrose fractions, samples were first digested with proteinase K (Sigma Aldrich, 1% of the sample volume; 100 mg/ml in 50 mM Tris-HCl pH=8.1, 10 mM CaCl 2 buffer) at 37°C for 45 min and shaking at 400 rpm. The digested samples were mixed with 1 volume of Phenol:Chloroform:Isoamyl alcohol (PanReac AppliChem, 25:24:1, v/v), vortexed and spun down 5 min at 20,000 g at 4°C. Supernatants were transferred into 3 volumes of 100% ethanol, 0.1 volumes of 3M NaOAc pH=5.2 and 1 µl of GlycoBlue, and precipitated at -20°C. Samples were pelleted for 30 min at 20,000 g and 4°C, washed once with 100% ethanol and another time with 70% ethanol, dried and resuspended in 30 µl H 2 O.
Fractions were reverse transcribed and analysed by RT-qPCR.
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