For polysome profiling of primary erythroid progenitor cells, whole bone marrow was lineage-depleted (CD3, CD5, B220, Gr1, CD11b) using microbeads and the autoMACS as described above and stained for Ter119 and CD71. Erythroid progenitor cells (lineagenegativeCD71highTer119intermediate/high) were sort-purified in pure FCS, cultured for 1 hours in IMDM with 20% FCS and 2mM L-glutamine at 37°C at humidified conditions. Cells (ca. 3×106) were then harvested, spun down and incubated with 100µg/ml of cycloheximide for 10 minutes at 37°C, washed with ice-cold PBS containing 100µg/ml of cycloheximide and lysed in 300ul 5mM Tris (pH7.4), 2.5mM MgCl2, 1.5mM KCl. Gradients were poured using a Biocomp Gradient Station. Polysomes were separated on a 10–50% linear sucrose gradient containing 20mM HEPES-KOH (pH 7.4). 5mM MgCl2, 100mM KCL, 2mM DTT and 100µh/ml cycloheximide and centrifuged at 36,000 rpm for 2 hours in a Beckman Coulter L8-M centrifuge with SW40Ti rotor. Gradients were fractionated using a Gilson FC-203B fractionator. Absorbance at 254nm was used to visualize the gradients using a BioRad EM-1 Econo UV monitor.
Fc 203b fractionator
Manufactured by Gilson
The FC-203B fractionator is a lab equipment product designed for the separation and collection of liquid samples. It provides precise fraction collection capabilities to facilitate analytical processes. The core function of the FC-203B is to enable the fractionation and automated gathering of specific liquid components from a sample.
Automatically generated - may contain errors
Lab products found in correlation
Gradient station, by BioComp Instruments (1 mentions)
L8 m centrifuge, by Beckman Coulter (1 mentions)
Em 1 econo uv monitor, by Bio-Rad (1 mentions)
Optima l 100 xp preparative ultracentrifuge, by Beckman Coulter (1 mentions)
Ja 25, by Beckman Coulter (1 mentions)
2 ml lysing matrix e tube, by MP Biomedicals (1 mentions)
Fastprep 24 instrument, by MP Biomedicals (1 mentions)
3 protocols using fc 203b fractionator
1
Polysome Profiling of Erythroid Progenitors
2
Fractionation and Purification of Cellular Organelles
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Cells were harvested at 3500 rpm in Heraeus Multifuge, and pellets were washed twice with cold H2O, resuspended in 100 mM Tris HCl (pH 9.4), 10 mM DTT, and 10 μg/ml nocodazole, and incubated for 20 min on ice. Cells were washed with ice-cold H2O, resuspended in spheroplasting buffer (1 M sorbitol, 50 mM Tris HCl [pH 7.5], 1 mM CaCl2, 1 mM MgCl2, 10 μg/ml nocodazole, 350 U lyticase L4025-Sigma) and incubated 30 min on an orbital platform at 4°C. Spheroplasts were sedimented in a Beckman Coulter JA25.50 at 6000 rpm for 6 min, gently washed with 1 M sorbitol, transferred to 1.5 ml tubes, and sedimented for 1 min at 1500 rcf and 4°C. Pellets were resuspended in 200 μl cold 0.4 M sorbitol and lysed on ice for 30 min by the addition of 700 μl lysis buffer (25 mM HEPES/KOH [pH 8], 50 mM KCl, 10 mM MgSO4, 0.25% Triton X-100, 1 mM PMSF, 3 mM DTT, 1 × complete EDTA-free protease inhibitors), supplemented with 100 μg/ml RNase A and 300 mM NaCl. Cell extracts were obtained by spinning the lysed spheroplasts at 12,000 rcf and 4°C for 5 min.
Cleared lysates (450 μl) were loaded on sucrose gradients prepared in Biocomp gradient station and sedimented in SW41 rotor (Beckman Optima L-100 XP Preparative Ultracentrifuge) at 18,000 rpm for 4 hr. Gradients were fractionated using Gilson FC203B fractionator, collecting 15 drops/fraction.
Cleared lysates (450 μl) were loaded on sucrose gradients prepared in Biocomp gradient station and sedimented in SW41 rotor (Beckman Optima L-100 XP Preparative Ultracentrifuge) at 18,000 rpm for 4 hr. Gradients were fractionated using Gilson FC203B fractionator, collecting 15 drops/fraction.
3
Isolation and Analysis of Bacterial Ribosomes
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50 ml of E. coli MG1655 was grown to an OD600 of 2.0, followed by rapid filtration and immediate freezing in liquid nitrogen. The cells were then resuspended in 1 ml of ice-cold 1× lysis buffer B [20 mM Tris–HCl, pH 7.5, 100 mM NH4Cl, 10 mM MgCl2, 1 mM DTT, 1 mM PMSF, 0.4% Triton X 100, 20 U/ml DNase I, 200 U/ml RNase-inhibitor] and lysed using a FastPrep-24 instrument (MP Biomedicals) and a 2 ml lysing matrix E tube (MP Biomedicals) for 15 s at 4 m/s. To remove insoluble debris and the beads, the lysate was cleared by centrifugation for 10 min at 4°C and 16 100 rcf. Of the cleared lysate, 10 μl was mixed with 1 ml TRIzol for the RNA input control. Fifteen A260 nm per ml of the cleared lysate were then layered on top of a linear 10–55% (w/v) sucrose gradient (in 1× lysis buffer B without DNase I or RNase inhibitor and with addition of 5 mM CaCl2), which was formed in an open-top polyclear ultracentrifugation tube (Seton Scientific) using the Gradient Station model 153. The gradient was centrifuged for 2.5 h at 4°C and 237 000 rcf (35 000 rpm) using an SW 40 Ti rotor, followed by automated fractionation into 20 fractions using an FC 203B fractionator (Gilson). RNA extraction was performed as for the glycerol gradient, except that the vortexing step was performed for 15 s and that DNase treatment of the purified RNA was skipped.
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