Ribosomes were fractionated as in [37] (link). Briefly, 100 ml of yeast in exponential growth phase were treated with 100 µg/ml of CHX for 10 min on ice. Cells were harvested, washed with 50 ml of cold water with CHX, resuspended in 1 ml of buffer A (20 mM Hepes, pH8.0, 50 mM KCl, 10 mM MgCl2, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, 100 µg/ml CHX, and protease inhibitor cocktail (Roche)) and pelleted. Cells were broken with 0.5 ml of glass beads in 0.5 ml of buffer A for 15 min at 4°C. The lysates were clarified by centrifugation at 14000 g for 10 min. 0.2 ml of lysates containing 3 mg of total protein was applied on a 12 ml 7–47% sucrose gradient in 20 mM Hepes, 50 mM KCl, 10 mM MgCl2, 100 µg/ml CHX and centrifuged for 150 min at 220000 g at 4°C. Fractions were collected using a UA/6 detector (ISCO, Inc.), precipitated with TCA and separated by SDS-PAGE.
Ua 6 detector
Manufactured by Teledyne
The UA-6 detector is a laboratory instrument designed to detect and measure the presence of specific particles or elements. It functions by utilizing various sensing technologies to identify and quantify the target analytes within a sample. The UA-6 detector provides accurate and reliable data to support scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Sw40ti rotor, by Beckman Coulter (2 mentions)
Nucleospin rna 2, by Macherey-Nagel (1 mentions)
Cycloheximide (chx), by Promega (1 mentions)
Rnasin plus, by Promega (1 mentions)
Foxy r1 fraction collector, by Teledyne (1 mentions)
Firefly luciferase mrna, by Promega (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Reliaprep mirna cell and tissue miniprep system, by Promega (1 mentions)
Tube piercer, by Brandel Inc (1 mentions)
Sw41ti rotor, by Beckman Coulter (1 mentions)
Minipuls 3, by Gilson (1 mentions)
Gradient fractionator, by Teledyne (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Rneasy plus micro kit, by Qiagen (1 mentions)
8 protocols using ua 6 detector
1
Ribosome Fractionation Protocol
2
Sucrose Gradient Fractionation of HeLa Cell Lysates
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Cytoplasmic lysates of HeLa cells were prepared and subjected to centrifugation through linear sucrose gradients (10-50% sucrose) essentially as described previously [51 (link)]. siRNA transfected cells (3x 10cm plates per condition) were rinsed and scraped in ice-cold PBS containing 100μg/mL cycloheximide. All subsequent steps were performed in ice-cold conditions. Cells were pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mMDTT, 5 mM MgCl2, 0.5% Nonidet-P40, 0.1 mg/ml cycloheximide, and 0.5 mg/ml heparin), incubated 10 min on ice and clarified by centrifugation for 10 min at 12000g. Approximately 400μL of supernatant was layered onto a 12-ml linear sucrose gradient (10-50% sucrose (w/v) in detergent free extraction buffer) and centrifuged at 4°C in an SW40Ti rotor (Beckman, Palo Alto, CA) at 35,000 rpm without brake for 120 min. The gradients were collected into 12 fractions (1ml each), and absorbance profiles at 260 nm were recorded (ISCO, UA-6 detector). 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropyl alcohol were added to the probes for overnight precipitation at −20°C. RNA was purified using total RNA isolation kit (Nucleospin RNA II, Macherey & Nagel) following the manufacturer's protocol. RNA concentration was determined, and the samples were stored at −80°C.
3
Polysome Profiling of Mammalian Cells
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MEFs were grown in glucose or for 20 h in galactose medium to 70–80% confluency. Before harvest, cells were incubated at 37°C for 15 min in medium containing 100 µg/ml cycloheximide (Sigma-Aldrich). Cells were washed and harvested in 4°C cold PBS supplemented with 100 µg/ml cycloheximide and pelleted at 21,000 g for 10 s at 4°C. Cells were lysed for 30 min on ice in buffer comprised of 20 mM Tris-HCl, pH 7.4, 30 mM KCl, 15 mM MgCl2, 0.5% Triton X-100 (vol/vol), 2 mM DTT, 1 mg/ml heparin, 100 µg/ml cycloheximide, 0.16 U/ml RNase inhibitor (RNasin Plus; Promega), and 1× EDTA-free protease cocktail. 3–4 mg lysate was applied on a continuous 7–47% sucrose gradient (mol weight/volume) and centrifuged at 97,658 g for 3 h at 4°C. Polysome fractions were obtained using the Foxy R1 Fraction Collector, and the polysome profile was detected in real-time using a UA-6 detector (Teledyne ISCO). Collected fractions were shock frozen in nitrogen and stored at −80°C. RNA extraction was performed with LS-TRIzol (Ambion) according to protocol, and monosome and polysome RNA fractions were pooled, followed by DNase treatment and 2.5 M LiCl precipitation at 4°C overnight. 0.5 ng Firefly luciferase mRNA (Promega) spike-in control was added before cDNA preparation.
4
Ribosome Profiling of Drosophila Heads
5
Polysome Fractionation by Sucrose Gradient
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Polysome fractions were prepared using sucrose gradient fractionation as previously described [32 (link)]. To prepare polysomes, 1.25 × 107 HepG2 cells were rinsed and scraped in ice-cold PBS containing cycloheximide (0.1 mg/mL). Subsequent steps were carried out in the cold. After pelleting by centrifugation at 500× g for 7 min, the cells were resuspended in extraction buffer (20 mm Tris-HCl, pH 8.0, 140 mm KCl, 0.5 mm DTT, 5 mm MgCl2, 0.5% Nonidet-P40, 0.1 mg/mL cycloheximide, and 0.5 mg/mL heparin) and incubated for 5 min on ice. Extracts were centrifuged for 10 min at 12,000× g. Approximately 0.5 mL of supernatant was layered onto a 12-mL linear sucrose gradient (10–50% sucrose (w/v) in 20 mm Tris-HCl, pH 8.0, 140 mm KCl, 0.5 mm DTT, 5 mm MgCl2, 0.1 mg/mL cycloheximide, and 0.5 mg/mL heparin) and centrifuged at 4 °C in an SW40Ti rotor (Beckman, Palo Alto, CA, USA) at 35,000 rpm without brake for 80 min (120 min for experiments examining the distribution of β-globin reporter mRNAs). The gradients were collected into 10–12 1-mL fractions, and absorbance profiles at 260 nm were recorded (ISCO, UA-6 detector). An amount of 0.1 volume of 3 m sodium acetate (pH 5.2) and 1 volume of isopropyl alcohol were added to the probes for overnight precipitation at −20 °C. RNA was purified using the ReliaPrepTM miRNA cell and tissue miniprep system (Promega, Madison, WI, USA).
6
Sucrose Density Gradient Analysis of Translational Complexes
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Transfected cells on a 100-mm dish were incubated with 100 μg/ml of CHX for 10 min before harvesting. Cell pellets were lysed in sucrose density gradient buffer (50 mM Tris-Cl pH 7.5, 10 mM MgCl2, 25 mM KCl, 0.1 mg/ml heparin, 50 U/ml RNase inhibitor) containing 0.5% NP-40 (v/v), 1 mM PMSF and 1 mM DTT at 4°C for 15 min with gentle rocking. After clarification by centrifugation, soluble extracts were loaded on top of 10–50% (w/v) or 10–40% (w/v) sucrose density gradient solution and centrifuged at 38 000 rpm for 2 or 5 h, respectively, in an SW41Ti rotor (Beckman Coulter). Using Minipuls 3 (Gilson) and tube piercer (Brandel), gradient samples were fraction-collected into 1.5 ml tubes while UV absorbance at 254 nm was monitored continuously with a UA-6 detector (ISCO). Protein samples were prepared from individual fractions by methanol/chloroform precipitation, resolved by SDS-PAGE and then analyzed by immunoblotting (see above).
7
Ribosome Fractionation on Sucrose Gradient
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Ribosomes were fractionated on a 12 ml 7-47% sucrose gradient as in (Panasenko and Collart 2012) (link).
Briefly, 100 ml of yeast in exponential growth phase were treated or not with 100µg ml -1 of CHX, harvested, washed with cold water, and resuspended in buffer A (20 mM HEPES, pH 8.0, 50 mM KCl, 10 mM MgCl2, 1% Triton X-100, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail) with or without 100 µg ml -1 of CHX. Cells were broken with 0.5 ml of glass beads in 0.5 ml of buffer A for 15min at 4°C. The lysates were clarified by centrifugation at 14,000 g for 10 min. 0.2 ml of lysates containing 2-3 mg of total protein was applied on a 12 ml 7-47% sucrose gradient in 20 mM HEPES, 50 mM KCl, 10 mM MgCl2 with or without 100 µg ml -1 of CHX and centrifuged for 150 min at 220,000 g at 4°C. Fractions were collected using a UA/6 detector (ISCO), precipitated with TCA and separated by SDS-PAGE. When indicated, polysomes were dissociated by treatment with 25 mM EDTA added instead of CHX, in the lysis buffer and in the gradient.
Briefly, 100 ml of yeast in exponential growth phase were treated or not with 100µg ml -1 of CHX, harvested, washed with cold water, and resuspended in buffer A (20 mM HEPES, pH 8.0, 50 mM KCl, 10 mM MgCl2, 1% Triton X-100, 1 mM DTT, 1 mM PMSF and protease inhibitor cocktail) with or without 100 µg ml -1 of CHX. Cells were broken with 0.5 ml of glass beads in 0.5 ml of buffer A for 15min at 4°C. The lysates were clarified by centrifugation at 14,000 g for 10 min. 0.2 ml of lysates containing 2-3 mg of total protein was applied on a 12 ml 7-47% sucrose gradient in 20 mM HEPES, 50 mM KCl, 10 mM MgCl2 with or without 100 µg ml -1 of CHX and centrifuged for 150 min at 220,000 g at 4°C. Fractions were collected using a UA/6 detector (ISCO), precipitated with TCA and separated by SDS-PAGE. When indicated, polysomes were dissociated by treatment with 25 mM EDTA added instead of CHX, in the lysis buffer and in the gradient.
8
Sucrose Gradient Fractionation of Polysomes
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Sucrose gradient fractionation of polysomes of K562 cells were performed as previously described (Floor and Doudna, 2016) . Briefly, 2 T75 flasks of K562 cells (Ctrl and KO) were grown to a 2~3X10 5 cells/ml confluency in the growth media. 100μg/ml cycloheximide was added into the growth medium and incubated at 37˙C for 5 min. The cells were then collected by centrifugation, and re-suspended PBS+100 in μg/ml cycloheximide. Cells were then pelleted again by centrifugation and lysed with three pellet-volumes ice cold hypotonic lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1% Triton X-100 and 100 μg/ml cycloheximide). After 10 min, cells were lysed on ice by ten strokes through a 26-gauge needle and nuclei were pelleted at 1,500× g for 5 min. Lysate from ~15 million cells was layered on top of triplicate 10-50% (w/v) sucrose gradients (20 mM HEPES:KOH pH 7.6, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 100 μg/ml cycloheximide) made using a Biocomp Instruments (Canada) gradient master. Gradients were centrifuged for 2 h at 36,000 RPM in a SW-41 rotor, punctured, and manually peak fractionated using real-time A260 monitoring with a Brandel (Gaithersburg, MD) gradient fractionator and ISCO (Lincoln, NE) UA-6 detector. Fractions were subjected to RNA prep with RNeasy Plus micro kit (Qiagen 74034) and a RT-PCR analysis with SYBR green (Biorad, 1725120).
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