To obtain crude lysates, cell cultures were washed once with PBS containing Cycloheximide (CHX; 100ug/ml), harvested by cell scraping and then lysed on ice using 20mM Tris HCl pH7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT with 1% Triton-X + Protease Inhibitors + RNase inhibitors + CHX (100ug/ml). Nuclei and debris were separated from crude lysate by brief centrifugation at 15,000xg at 4°C. Sucrose gradients (10%–50%) were prepared in 20mM Tris HCl pH7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT + RNase inhibitors + CHX (100ug/ml) using a Biocomp Model 108 gradient master. Crude cellular lysates were then loaded onto gradients and separated by centrifugation at 110,000xg, 3 hours at 4°C and fractionated into 0.5mL aliquots using a Biocomp Model 152 Piston Fractionator. Polysome fractions (typically fractions #10 through #20) were pooled and RNA extraction/purification was performed for the preparation of sequencing libraries.
Model 108 gradient master
Manufactured by BioComp Instruments
Sourced in Canada
The Model 108 gradient master is a laboratory instrument designed for creating linear gradients. It can generate gradients of various solutions, such as density or concentration, within a container.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using model 108 gradient master
1
Polysome Profiling for Ribosome Dynamics
2
Polysome Profiling of Cycloheximide-Treated Cells
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The cells were treated with 100-µg/mL cycloheximide diluted in culture medium (Sigma, St. Louis, MO, USA) (10 min, 37 °C), harvested, washed twice (100-µg/mL cycloheximide in PBS) and centrifuged (700× g, 5 min). The cell pellet was incubated with lysis buffer (15-mM Tris-HCl, pH 7.4; 15-mM MgCl2; 300-mM NaCl; 100-µg/mL cycloheximide and 1% Triton X-100) for 10 min on ice and centrifuged (12,000× g, 10 min, 4 °C), and the supernatant was loaded on a sucrose density gradient (10–50%), which was prepared with a model 108 Gradient Master from BioComp (Fredericton, NB, Canada). Then, the samples were centrifuged at 150,000× g (SW40 rotor, HIMAC CP80 WX HITACHI, Tokyo, Japan) for 2.5 h at 4 °C. The sucrose fractions were collected using an ISCO system (ISCO Model 160 Gradient Former Foxy Jr. Fraction Collector, Teledyne Isco, St. Lincoln, NE, USA) with an associated UV detector (Teledyne Isco, St. Lincoln, NE, USA). The absorbance was recorded at 275 nm for use in determining the polysome profile.
3
Polysome Profiling for Ribosome Dynamics
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