Ua 6 absorbance detector

Cell lysates of HEK293T and A549 cells for sucrose gradient centrifugation were prepared in polysome extraction buffer (20 mM HEPES at pH 7.6, 100 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol [DTT], 100 μg/ml cycloheximide, 100 U/ml RNAse inhibitor [Thermo Scientific] and Halt Protease and Phosphatase Inhibitor Cocktail [Thermo Scientific]). Extracts were incubated on ice for 20 min, and insoluble material was pelleted by centrifugation at 12000 rpm for 10 min. The resulting supernatant extracts were loaded onto a 10–50% (w/v) sucrose gradient and ultracentrifuged at 30000 rpm for 3 hours at 4 °C in a Surespin 630 rotor (Sorvall). Following centrifugation, the gradients were fractionated using a fraction collector (Brandel), and their quality was monitored at 254 nm using a UA-6 Absorbance Detector (Isco). Proteins from sucrose gradient fractions were precipitated with 10% (v/v) TCA, separated by SDS-PAGE, and transferred to PVDF membranes for immunoblotting.

Fractionation of polyribosomes was done essentially as described before (9 (link),10 (link)) using 10–50% (17 000 rpm., 18 h) and/or 10–30% (20 000 rpm., 18 h) sucrose gradients and a Beckman SW32.1 rotor. All procedures were performed at 4°C. Yeast cells from 50 ml of log phase culture were pelleted, treated for 10 min with 100 μg/ml cycloheximide and repelleted. Cell extracts were made by glass bead cell disruption (3–5 cycles of 1 min each), with intermittent cooling on ice. The following buffer was used: 100 mM KCl, 2.5 mM magnesium acetate, 20 mM HEPES·KOH, pH 7.4, 14.4 mM β-mercaptoethanol, 100 μg/ml cycloheximide. Cell debris was removed by centrifugation at 7000 rpm for 8 min and polyribosomes were resolved by sucrose density gradient centrifugation as indicated. Gradients were collected using the ISCO Programmable Density Gradient System with continuous monitoring at 254 nm using an ISCO UA-6 absorbance detector. Analysis of ratios of 80S monosomes to polyribosomes was done essentially as before (10 (link)). Fractionation of cell extracts using formaldehyde cross-linking was done as described by Nielsen et al. (18 (link)). For western blotting, proteins collected from sucrose gradient fractions were solubilized in sodium dodecyl sulfate (SDS) polyacrylamide sample buffer for 10 min at 95°C, chilled on ice for 5 min and loaded onto polyacrylamide gel.

The polysome analysis was patterned on the method of Zhao et al. (32 (link)). Sucrose density gradients were prepared using five layers of buffered sucrose (50 mm Tris, 50 mm ammonium acetate, 12 mm MgCl₂, pH 7.0, and either 7%, 21%, 33%, 47%, or 60% sucrose). 1 ml of each mixture was placed in a centrifuge tube from high density (60%) at the bottom to low density (7%) at the top. This step gradient was stored at 4 °C overnight to allow formation of a continuous gradient. The next morning, flash-frozen liver tissue was homogenized in lysis buffer (15 mm MgCl₂, 200 mm KCl, 1% Triton x-100, 100 μg/ml cycloheximide, 2 mm DTT, 0.1% diethyl pyrocarbonate, pH 7.4) at 6 m/s for 60 s in a MP-Biomedicals FastPrep®-24. The homogenate was centrifuged to clarify (14,000 g, 5 min, 4 °C). Approximately 1.2 mg of total protein were loaded onto each gradient. The sample was then separated within the sucrose gradient using high speed centrifugation (99,526 g, 4 h, 4 °C) in a Beckman Coulter Optima™ l-100 XP. Polysomes were analyzed using an Isco UA-6 Absorbance Detector at 254 nm. Six mice from AL and six from DR were run in duplicate for assessment of polysome profiles.

NCM460 cells were treated with 1.5 mM aspirin for 24 hours. At 70% of confluence, cells were treated with 100 μg/ml Cycloheximide (CHX) for 15 min. Cells were washed twice on ice in PBS + 100 μg/ml CHX, then resuspendend in a LSB buffer [20 mM Tris HCl, 10 μM NaCl, 3 mM MgCl2, 100 μg/ml CHX, 0.04 U/μl RiboLock RNAsi inhibitor (Thermo Scientific, Waltham, MA, USA) protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland)] and lysate on ice for 10 min adding a detergent buffer [0.3% Triton N101 50 mM Sucrose, 100 μg/ml CHX, 0.04 U/μl RiboLock RNAsi inhibitor (Thermo Scientific)]. The lysate was centrifuged at 14000 rcf for 10 min at 4°C. Ribosomes were then separated on chilled 15%–50% Sucrose Gradient (in LSB buffer by ultracentrifugation at 160000 × g for 2 hours at 4°C). The polysome profile was monitored at 254 nm (0.2 O.D. sensibility) and fractionated (at 10×, 10% Tris-Pump power) using an ISCO gradient fractionator system interfaced to an UA-6 absorbance detector (Teledyne Isco, Lincon, NE, USA). The data collected were digitally converted by using Minilab 1008 (Measuring Computing, Norton, MA, USA) and TracedDaq software (Measuring Computing, Norton, MA, USA), acquiring data in a differential mode at +/− 4 V and 4Hz.

Polysomes were isolated by the method described by Yang et al. [73 (link)]. Briefly, about 1 g of pulverized NB or SR86 shoots in liquid nitrogen was transferred to a 15-mL nuclease-free centrifuge tube with 5 mL ice-cold polysome extraction buffer (PEB) [100 mM Tris-HCl (pH 8.0), 40 mM KCl, 20 mM MgCl₂, 2% (V/V) polyoxyethylene-10-tridecyl ether, 0.2% (W/V) deoxycholic acid, 1 mM DTT, 100 μg mL^− 1 cycloheximide and 10 U mL^− 1 DNaseI] and mixed well. The suspension was then centrifuged at 4 °C with a speed of 5000 g for 10 min, followed by another 20,000 g centrifugation at 4 °C for 10 min in a new 15-mL tube. The supernatant was subjected to polysome profile analysis and RPF isolation.
To perform profile analysis, the isolated polysome sample was loaded on a 15–60% (W/V) sucrose gradient in a polypropylene centrifuge tube (13 × 51 mm, BECKMAN, USA) and then centrifuged in a SW-55 rotor (BECKMAN, USA) at 4 °C with a speed of 170,000 g for 1.5 h. Fractionation, absorbance assay and data acquisition of the resulting gradient were carried out using a gradient fractionator system (BRANDEL, USA) with a UA-6 absorbance detector (TELEDYNE ISCO, USA).