Sucrose solutions were prepared in polysome gradient buffer [10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 μg/mL cycloheximide, 5 mM DTT, and 20 U/mL SUPERase_In (Ambion)]. Sucrose density gradients [15–45% (wt/vol)] were freshly made in SW41 ultracentrifuge tubes (Fisher) using a BioComp Gradient Master (BioComp) according to the manufacturer's instructions. Puromycin-treated cell lysate was loaded onto sucrose gradients, followed by centrifugation for 100 min at 38,000 rpm, 4 °C, in an SW41 rotor. Separated samples were fractionated at 0.375 mL/min by using a fractionation system (Isco) that continually monitored OD254 values. Fractions were collected into tubes at 1-min intervals.
Fractionation system
Manufactured by Teledyne
Sourced in United States
The Fractionation system is a lab equipment product that separates a mixture into its constituent parts or fractions based on differences in physical or chemical properties, such as boiling point, molecular weight, or polarity. The core function of this system is to facilitate the separation and purification of complex mixtures for analytical or preparative purposes.
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Lab products found in correlation
7 protocols using fractionation system
1
Sucrose Density Gradient Fractionation
2
Sucrose Density Gradient Fractionation
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Sucrose solutions were prepared in polysome gradient buffer [10 mM HEPES, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 μg/mL cycloheximide, 5 mM DTT, and 20 U/mL SUPERase_In (Ambion)]. Sucrose density gradients [15–45% (wt/vol)] were freshly made in SW41 ultracentrifuge tubes (Fisher) using a BioComp Gradient Master (BioComp) according to the manufacturer's instructions. Puromycin-treated cell lysate was loaded onto sucrose gradients, followed by centrifugation for 100 min at 38,000 rpm, 4 °C, in an SW41 rotor. Separated samples were fractionated at 0.375 mL/min by using a fractionation system (Isco) that continually monitored OD254 values. Fractions were collected into tubes at 1-min intervals.
3
Polysome Fractionation and Analysis
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Cells were first treated with cycloheximide (100 μg/mL) for 3 min at 37°C to immobilize the translating ribosomes. After ice-cold PBS solution wash, cells were then harvested by ice-cold polysome lysis buffer [10 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 μg/mL cycloheximide, and 2% (vol/vol) Triton X-100]. After centrifugation at 12,000 × g for 10 min at 4°C, the supernatant was subjected to sucrose gradient sedimentation. Briefly, sucrose solutions were prepared in polysome gradient buffer (10 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2, 100 μg/mL cycloheximide). Sucrose density gradients [15–45% (wt/vol)] were freshly made in SW41 ultracentrifuge tubes (Fisher) using a BioComp Gradient Master (BioComp) according to the manufacturer's instructions. Approximately 650 μL of cell lysate was loaded onto sucrose gradients, followed by centrifugation for 100 min at 38,000 rpm, 4°C, in an SW41 rotor. Separated samples were fractionated at 0.375 mL/min by using a fractionation system (Isco) that continually monitored OD254 values. Fractions were collected into tubes at 1-min intervals.
4
Polysome Profiling of Treated Cells
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Either MDA-MB231 or HCC1806 cells were treated with 100 μg/mL cycloheximide in growth media for 5 minutes at 37 °C. The cells were washed with ice-cold 1x PBS containing 100 μg/mL cycloheximide prior to harvesting by scraping. The cells were lysed in polysome lysis buffer (20 mM Tris pH 7.26, 130 mM KCl, 10 mM MgCl2, 0.5% NP-40, 0.2 mg/mL heparin, 200 U/mL RNasin, 2.5 mM DTT, 1x HALT, 100 μg/mL cycloheximide, 0.5% sodium deoxycholate) for 20 minutes on ice prior to clarification at 8000 g for 10 minutes at 4 °C. The absorbance at 260 nm was determined for each lysate. An equal number of A260 units for each lysate was overlaid on a 10–50% sucrose gradient (10 mM Tris pH 7.26, 60 mM KCl, 10 mM MgCl2, 2.5 mM DTT, 0.2 mg/mL heparin, 10 μg/mL cycloheximide). The gradients were subjected to centrifugation at 30,000 RPM for 3 hours at 4 °C. The absorbance at 254 nm was measured along the gradient using a fractionation system (Teledyne ISCO, Lincoln, NE USA).
5
Polysome Profiling of Inflorescence Samples
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Three induced inflorescence samples of WT and drmy1 in ap1 cal AP1-GR background were collected as described above, and polysomes were extracted as previously described138 (link). Briefly, samples were ground in liquid nitrogen, mixed with an extraction buffer (0.2 M Tris pH 9.0, 0.2 M KCl, 0.025 M EGTA, 0.035 M MgCl2, 1% (w/v) Brij-35, 1% (v/v) Triton X-100, 1% (v/v) Igepal CA-630, 1% (v/v) Tween-20, 1% (w/v) Sodium deoxycholate, 1% (v/v) Polyoxyethylene 10 tridecyl ether, 5 mM Dithiothreitol, 1 mM Phenylmethylsulfonyl fluoride, 100 μg/ml cycloheximide, 100 μg/ml chloramphenicol, 40 U/ml RNasin, 0.5 mg/ml Heparin), and let sit on ice for 10 min. Samples were centrifuged at 4°C 4,000 g for 5 min, supernatant was transferred to a new tube, centrifuged at 4°C 16,000 g for 15 min, and supernatant was filtered through Miracloth.
Polysome extracts were profiled as previously described139 (link). Briefly, samples were loaded onto 15%-45% sucrose density gradients and centrifuged at 4°C 38,000 rpm in a SW41 rotor. Separated samples were fractionated at a rate of 0.375 mL/min in an Isco fractionation system, and absorbance at 254 nm was recorded.
Polysome extracts were profiled as previously described139 (link). Briefly, samples were loaded onto 15%-45% sucrose density gradients and centrifuged at 4°C 38,000 rpm in a SW41 rotor. Separated samples were fractionated at a rate of 0.375 mL/min in an Isco fractionation system, and absorbance at 254 nm was recorded.
6
Polysome Profiling of Treated Cells
7
Polysome Profiling of Cycloheximide-Treated Cells
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Huh-7 and HCC-LM3 cells were pre-treated with 200 µM cycloheximide (Sigma, USA) for 5min at 37°C and washed with ice-cold PBS containing 200 µM cycloheximide.
Cells were then lysed with polysome lysis buffer for 30 min on ice. After centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was loaded onto 10 mL continuous 15-50% sucrose gradients buffer containing 50 U/ml RNase inhibitor. The samples were centrifuged at 4°C for 3 h at 100,000 g by using Avanti J-30XP (Beckman, USA), and the fractions were collected using a Brandel Fractionation System (USA) and an Isco UA-6 ultraviolet detector (USA) was used to produce polysome profiles for gradients.
Extraction and transcription of total RNA from each fraction and RT-PCR was conducted as showing above. GAPDH served as positive control.
Cells were then lysed with polysome lysis buffer for 30 min on ice. After centrifugation at 14,000 rpm for 10 min at 4°C, the supernatant was loaded onto 10 mL continuous 15-50% sucrose gradients buffer containing 50 U/ml RNase inhibitor. The samples were centrifuged at 4°C for 3 h at 100,000 g by using Avanti J-30XP (Beckman, USA), and the fractions were collected using a Brandel Fractionation System (USA) and an Isco UA-6 ultraviolet detector (USA) was used to produce polysome profiles for gradients.
Extraction and transcription of total RNA from each fraction and RT-PCR was conducted as showing above. GAPDH served as positive control.
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