Five–fifty percent wt/vol linear sucrose gradients were freshly casted on SW41 ultracentrifuge tubes (Beckmann) using the Gradient master (BioComp instruments) following the manufacturer’s instructions. Forty-eight hours post transfection, H1299 cells (with 80% confluency) were treated with cycloheximide 100 µg/ml for 5 min at 37 °C and then washed twice with 1× PBS (Dulbecco modified PBS, GIBCO) containing cycloheximide 100 µg/ml. Cells were then scrapped and lysed with polysome lysis buffer (100 mM KCL, 50 mM HEPES-KOH, 5 mM MgCl2, 0.1% NP-40, 1 mM DTT, cycloheximide 100 µg/ml, pH 7.4). Lysates were then loaded on a sucrose gradient and centrifuged at 222228×g for 2 h at 4 °C in a SW41 rotor. Samples were then fractionated using Foxy R1 fraction collector (Teledyne ISCO) at 0.5 min intervals35 ,36 (link). RNA purifications from fractions were performed using Trizol-LS reagent (Invitrogen) combined with ethanol precipitation. Reverse transcription for the target mRNAs were carried out using equal volume of DNA free RNA from fractions and then normalised with actin levels.
Foxy r1 fraction collector
Manufactured by Teledyne
Sourced in Germany
The Foxy R1 fraction collector is a laboratory instrument designed for the automated collection of fractions during chromatographic separations. It features a peristaltic pump that precisely dispenses samples into collection vessels, ensuring accurate and reproducible fraction collection.
Automatically generated - may contain errors
Lab products found in correlation
Sw41 ultracentrifuge tube, by Beckman Coulter (3 mentions)
Dulbecco modified pbs, by Thermo Fisher Scientific (3 mentions)
Gradient master, by BioComp Instruments (3 mentions)
Cycloheximide (chx), by Promega (2 mentions)
Rnasin plus, by Promega (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (1 mentions)
Ua 6 detector, by Teledyne (1 mentions)
Firefly luciferase mrna, by Promega (1 mentions)
L 7455, by Hitachi (1 mentions)
Ribonuclease inhibitor, by Thermo Fisher Scientific (1 mentions)
Sw40ti rotor, by Beckman Coulter (1 mentions)
Edta free, by Roche (1 mentions)
Isco ua 6 absorbance detector, by Teledyne (1 mentions)
717 plus autosampler, by Waters Corporation (1 mentions)
Plrp s column, by Agilent Technologies (1 mentions)
Lichrospher 100, by Merck Group (1 mentions)
Microtof q 2, by Bruker (1 mentions)
12 protocols using foxy r1 fraction collector
1
Polysome Profiling of Cycloheximide-Treated Cells
2
Polysome Profiling of Stalled Ribosomes
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Cells were harvested at 70–80% of confluence. Polysomes were stalled by treating the cells with 100 µg/ml cycloheximide (Sigma-Aldrich) for 15 min. Cells were washed and scraped in PBS containing cycloheximide at 4°C. After pelleting cells at 21,000 g for 15 s, the pellet was lysed in buffer containing 20 mM Tris-HCl, pH 7.4, 30 mM KCl, 0.5% Triton X-100, 2 mM DTT, 1 mg/ml heparin, 100 µg/ml cycloheximide, and 0.16 U/ml RNase inhibitor (RNasin Plus; Promega). The lysates were then loaded onto a 7–47% (mol wt/vol) continuous sucrose gradient and centrifuged at 97,658 g for 3 h at 4°C in a rotor (SW41; Beckman Coulter). For polysome profiling, fractions were monitored at 254 nm using a UA-6 detector and collected with a density gradient fractionation system (Foxy R1 Fraction Collector; Teledyne ISCO). For Western blot analysis, the same volume of samples from each fraction was loaded on the SDS-PAGE.
3
Polysome Profiling of E2F1 mRNA
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5–50% (w/v) linear sucrose gradients were freshly casted on SW41 ultracentrifuge tubes (Beckmann) using the Gradient master (BioComp instruments) following the manufacturer's instructions. 48 h post transfection, H1299 cells (with 80% confluency) were treated with cycloheximide 100 μg/ml for 5 min at 37°C and washed twice with 1 × PBS (Dulbecco modified PBS, GIBCO) containing cycloheximide 100 μg/ml. Cells were lysed with polysome lysis buffer (100 mM KCl, 50 mM HEPES–KOH, 5 mM MgCl2, 0.1% NP-40, 1 mM DTT, cycloheximide 100 μg/ml, pH 7.4) and clear lysate was loaded on a sucrose gradient and centrifuged at 222 228 × g for 2 h at 4°C in a SW41 rotor. Samples were fractionated using Foxy R1 fraction collector (Teledyne ISCO) at 0.5 min intervals. Collected fractions were then pooled accordingly (free pool, 40S/60S, monosome and polysome fractions), concentrated using Amicon® Ultra-15 Centrifugal Filter Units (Merckmillipore) and subjected to Western and RT-qPCR analysis. RNA purifications from fractions were performed using ethanol precipitation combined with RNeasy Mini Kit (Qiagen). RT and qPCR were performed as described above using primers described in Supplementary Table S1 . The ratio of the fold enrichment of E2F1 mRNA levels in the stress induced cells (GAr) to the normal cells (EV) were plotted, actin levels were used for the normalization.
4
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.
5
Profiling Translational Regulation via Sucrose Gradients
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Five–fifty percent (w/v) linear sucrose gradients were freshly casted on SW41 ultracentrifuge tubes (Beckmann) using the Gradient master (BioComp instruments) following the manufacturer's instructions. Forty-eight hours post transfection, H1299 cells (with 80% confluency) were treated with cycloheximide 100 μg/ml for 5 min at 37°C and washed twice with 1× PBS (Dulbecco modified PBS, GIBCO) containing cycloheximide 100 μg/ml. Cells were then scraped and lysed with polysome lysis buffer (100 mM KCl, 50 mM HEPES–KOH, 5 mM MgCl2, 0.1% NP-40, 1 mM DTT, cycloheximide 100 μg/ml, pH 7.4). Lysates were loaded on a sucrose gradient and centrifuged at 222 228 × g for 2 h at 4°C in a SW41 rotor. Samples were fractionated using Foxy R1 fraction collector (Teledyne ISCO) at 0.5 min intervals (30 (link)). RNA purifications from fractions were performed using ethanol precipitation combined with RNeasy Mini Kit (Qiagen). RT and qRT-PCR were performed as described above using primers described in Supplementary Table S2 . The relative distribution of target mRNA was calculated using fraction 1 as reference according to Panda et al. (31 ).
6
HPLC Purification of Protein Samples
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Clear supernatant from D-75 and D-2189 were collected as described above. The 3 kDa Amicon Ultra – 15 Centrifugal filtered supernatants were lyophilized and reconstituted in double-distilled water (DDW) and were analyzed using a Merck Hitachi Lachrom HPLC system controlled by EZChrom Elite software. For analytical separation we used a Kinetex® 5 μm EVO C18 100 Å, LC Column 150 × 4.6 mm and an injection volume of 5–10 μL. For production of larger amounts we used C-18 semi-preparative column (Kinetex® 5 μm EVO C18 100 Å, AXIA Packed LC Column 150 × 21.2 mm) at a flow rate of 9.9 ml/min and an injection volume of 100–500 μL. In both cases, the RP-HPLC method employed linear gradient elution using two mobile phase buffers: Buffer A, 0.1%TFA in acetonitrile, and Buffer B, 0.1%TFA in water. The gradient was as follows: 0–3 min, 100% Buffer B, 3–17 min, 100–85% Buffer B, 17–18 min, 85 to 5% buffer B, after which the column was re-equilibrated to initial conditions over a period of 7 min. The UV absorbance signal was collected by a Hitachi L- 7455 diode array detector in the range 200–400 nm. The chromatograms at wavelengths of 220 and 280 nm were used to detect the peaks. Fractions were collected using a Foxy R1 fraction collector (Teledyne Isco) and dried for further characterization.
7
Ribosome-bound mRNA Isolation by Polysome Separation
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Isolation of ribosome-bound mRNA by polysome separation was performed as described previously with minor modifications (Silvera et al. 2017 (link)). Briefly, MCF7 cells were seeded 48 h prior to treatment, and cells were treated with 100 µg/mL cycloheximide for 10 min at 37°C, trypsinized, and collected in ice-cold PBS containing protease inhibitor cocktail and EDTA-free (Roche Diagnostics). All subsequent steps contained 100 µg/mL cycloheximide. Cells were resuspended in low-salt buffer (LSB; 20 mM Tris at pH 7.4–7.5, 10 mM NaCl, 3 mM MgCl2, ribonuclease inhibitor [Thermo Scientific]) and incubated for 3–5 min on ice. Detergent buffer (LSB with 1.2% Triton X-100, 0.2 M sucrose) was then added, and cells were lysed with 15–20 strokes in a sterilized Dounce homogenizer at 4°C. Lysates were cleared by microfuge centrifugation at maximum speed for 5 min, and supernatant was combined with 100 µL of heparin buffer (LSB with 10 mg/mL heparin, 1.5 M NaCl) and then layered on a 15%–50% sucrose gradient in LSB using equal OD260 units of samples. Gradients were centrifuged at 36,000 rpm for 2 h in a SW40Ti rotor (Beckman Coulter), and polysome profiles were done at UV absorbance 254 nm by continuous flow cell monitoring and collected using an Isco UA-6 absorbance detector (Teledyne ISCO) and with a Foxy R1 fraction collector (Teledyne ISCO) at 1.5 mL/min.
8
Peptide Quantification by HPLC
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Lyophilized reaction products were dissolved in 60% mobile phase in concentration 10 mg of dry mass per ml. The mixture was centrifuged at 4°С, 16,000g 15 min, supernatant was collected and diluted with mobile phase A to 30% mobile phase concentration.
Purification was performed on Azura HPLC system (P6.1L, DAD 2.1L, AS 6.1L, CT 2.1) (Knauer, Germany) with Foxy R1 fraction collector (Teledyne ISCO, USA). ACE 3C18-300 4.6х150 mm column (Advanced Chromatography Technologies, UK) was equipped. Chromatography conditions: column flow 1 mL/min, gradient elution from 0% to 30% in 3 min and from 30% to 100% B in 17 min (A: 0.1% (v/v) formic acid in water, B: 0.1% (v/v) formic acid in ACN), column temperature 55°C, injection volume 1000 μL. Fractions were collected with 1AU threshold on 280 nm wavelength.
100 μL of every hydrolyzed sample (n=4) were injected. Target peak area was converted into peptide dry mass with molar extinction of Trp, Tyr and Cys under denaturing conditions [18] (link).
Purification was performed on Azura HPLC system (P6.1L, DAD 2.1L, AS 6.1L, CT 2.1) (Knauer, Germany) with Foxy R1 fraction collector (Teledyne ISCO, USA). ACE 3C18-300 4.6х150 mm column (Advanced Chromatography Technologies, UK) was equipped. Chromatography conditions: column flow 1 mL/min, gradient elution from 0% to 30% in 3 min and from 30% to 100% B in 17 min (A: 0.1% (v/v) formic acid in water, B: 0.1% (v/v) formic acid in ACN), column temperature 55°C, injection volume 1000 μL. Fractions were collected with 1AU threshold on 280 nm wavelength.
100 μL of every hydrolyzed sample (n=4) were injected. Target peak area was converted into peptide dry mass with molar extinction of Trp, Tyr and Cys under denaturing conditions [18] (link).
9
Peptide Purification by HPLC
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The peptide was purified in four batches by high performance liquid chromatography (HPLC) on a VWR-Hitachi LaChrom Elite instrument (VWR, Darmstadt, Germany) equipped with a semi-preparative PLRP-S column (325 mm × 30 mm, 30 nm pore diameter, 8 µm particle size; Agilent, Santa Clara, CA, USA), a VWR-Hitachi L2400 UV detector (VWR, Darmstadt, Germany) and a Foxy R1 fraction collector (Teledyne ISCO, Lincoln, NE, USA). A flow rate of 6 mL min -1 and the following gradient (solvent A: water, 0.1% formic acid (FA), solvent B: acetonitrile, 0.1% FA) was applied: 0 min: 5% B, Elite instrument (VWR, Darmstadt, Germany) equipped with a 717 plus autosampler (Waters, Milford, MA, USA), an analytical PLRP-S column (150 mm × 4.6 mm, 30 nm pore diameter, 8 µm particle size; Agilent, Santa Clara, CA, USA), a VWR-Hitachi L2400 UV detector (VWR, Darmstadt, Germany) and an expressionL cms MS device (Advion, Harlow, UK). A flow rate of 1 mL min -1 and the following gradient (solvent A: water, 0.1% FA, solvent B: acetonitrile, 0.1% FA) was applied: 0.0 min: 5% B, 2.5 min: 5% B, 12.5 min: 60% B, 13.5 min: 95% B, 16.0 min: 95% B, 17.0 min: 5% B, 18.5 min: 5% B. A dead time of 2.0 min was observed. TC(4,8) eluted after retention time of t R = 9.8 min. Fractions deemed sufficiently pure were combined and lyophilized. The peptide was obtained as a white fluffy solid (17.8 mg, 8.65 µmol, 5%).
10
Isoflavone Analysis by HPLC-MS
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Chromatographic determination was performed on VWR Hitachi Chromaster 600 chromatograph (Merck) with a pump (5160), a degasser, a thermostat (5310), an autosampler (5260), a DAD detector (5430) and EZChrom Elite software. The extracts were analyzed on C18 reversed-phase column LiChrospher 100 (Merck) (25 cm × 4.0 mm i.d., 5 μm particle size), at a temperature of 30 °C. A 20 µL portion of the sample was injected. A mixture of acetonitrile (A) and water (B) with 0.025% of trifluoroacetic acid was used as a mobile phase. The gradient program was as follows: 0–10 min 20% A, 80% B; 10–30 min 25% A, 75% B; 30–60 min 35% A, 65% B. The flow rate of the eluent was 1.5 mL·min−1. The data were collected in a wavelength range from 200 to 400 nm. The chromatographic fractions eluted at a retention time characteristic for the investigated isoflavones were collected using a Foxy R1 fraction collector (Teledyne Isco, Lincoln, NE, USA) and analyzed by direct-injection mass spectrometry with electrospray ionization (micrOTOF-Q II, Bruker Daltonics, Bremen, Germany) using Compass DataAnalysis software Version 4.1.
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