RiboPuromycylation

Here is the protocol: “Ribopuromycylation”:   Whole blood (4 ml) was taken from healthy volunteers and collected in heparinized BD vacutainers. This protocol was approved by the Institute’s Human Ethics Committee (IHEC No: 18-28102016). The whole blood was then transferred to an open syringe without piston and closed at the tip. The syringe was centrifuged at 800 x g for 20 minutes at 4 ºC. The resulting plasma was discarded. After opening the syringe at the tip, about two-thirds of the sedimented erythrocytes were allowed to carefully drop out of the syringe. Special attention was paid not to disturb the WBC layer above the erythrocytes. The erythrocyte pellet was washed thrice with RBC wash buffer containing 21 mM Tris, 4.7 mM Potassium chloride (KCl), 2 mM Calcium chloride (CaCl2), 140.5 mM Sodium chloride (NaCl), 1.2 mM Magnesium sulphate (MgSO4), 5.5 mM Glucose, and 0.5% Bovine serum albumin (BSA). In between each wash, the contents were centrifuged at 500 x g for 10 minutes at 4 ºC. The RBC pellet obtained after centrifugation was depleted of CD71+ cells using CD71 MACS MicroBeads (Miltenyi Biotech). 10^7-10^8 cells were resuspended in 80 µL of MACS buffer (degassed) containing 0.5% BSA, 2 mM EDTA in 1X PBS. 20 µL of CD71 MicroBeads was added per 10^7 cells. The contents were mixed well and incubated at 4 °C for 15 minutes. The cells were washed by adding 1-2 mL of buffer per 10^7 cells. The cells were resuspended in 500 µL buffer. Meanwhile, LD columns (Miltenyi Biotech) were prepared by rinsing with 2 mL buffer. The cell suspension was applied to the column and allowed to pass through under the influence of gravity alone. The unlabelled cells were collected and the column was washed with 2x1 mL of buffer. The total effluent, i.e., the CD71- population was collected. Alternatively, FACS based sorting can be done using CD71 antibody (SC-32272 from Santa Cruz Biotechnology). Approximately 106 cells were washed with FACS buffer containing 5% BSA (range 1-5%) and 1% sodium azide in 1X PBS. 1μg of CD71 antibody added per 106 cells and incubated at 4°C overnight. The cells were then washed thrice with FACS buffer at 300g for 3 minutes. The fluorescently labelled secondary antibody was added at a dilution of 1:500 (in FACS buffer) and incubated in dark on ice for 45 minutes. After staining, cells were resuspended in complete RPMI 1640 media and CD71- cells were selectively sorted using BD FACSAria Fusion flow cytometer and sorter.  The isolated mature erythrocytes were treated with 91 μM puromycin (Sigma-Aldrich) for 4 hours at 37°C (David et al, 2012). Cells were then smeared on glass slides by placing a drop of mature erythrocytes and dragging another slide at a 45-degree angle. These slides were air-dried. They were fixed with ice-cold methanol for 15 seconds at room temperature and washed thrice with PBS. Blocking was done with buffer containing 1% BSA, 22.52 mg/ml glycine, 0.1% Tween-20 in PBS for 30 minutes. Cells were then incubated overnight at 4°C with anti-puromycin antibody followed by secondary antibody conjugated Alexa Fluor 488. After three washes, the coverslip was mounted on the slide using DuoLink in situ mounting medium (Sigma-Aldrich). Slides were imaged using 100X objective (Super Apo Chromatic lens with numerical aperture 1.35) in a confocal microscope (Olympus FLUOVIEW FV3000). The imaging medium was Olympus IMMOIL-F30CC (low autofluorescence immersion oil Type F). A high sensitivity spectral detector (GaAsP) was used to visualize the fluorescence. FLUOVIEW FV31S-SW software was used to acquire images.    “Isolation of ribosomes using sucrose cushion”:  Ribosomes were isolated from mature erythrocytes using sucrose cushion as described previously (Stéphane Belin et al, 2010). About 2 ml of mature erythrocyte pellet was resuspended in buffer containing 250 mM sucrose, 250 mM KCl, 5 mM MgCl2, 50 mM HEPES-KOH (pH 7.4), and 100 µg/ml cycloheximide. Since erythrocytes have higher cell density in a given volume as compared to other mammalian cells, the volume of buffer used was five times the volume of the cell pellet. This was done to obtain a homogeneous mixture. Cell lysis was performed by adding NP-40 (0.7% final concentration). Lysate was centrifuged at 12500 g for 10 minutes at 4 °C to pellet any organelles present.     11 ml of the supernatant was layered on 1 ml of sucrose cushion (1 M sucrose, 0.5 M KCl, 5 mM MgCl2, 50 mM HEPES-KOH (pH 7.4), and 100 µg/ml cycloheximide) in a polycarbonate tube (13.5 ml capacity). Tubes were centrifuged at 45000 rpm (equivalent to 183254 RCFmax; TI 50 rotor and L8-60M ultracentrifuge, Beckman Coulter) for 4 hours at 4° C. Ribosomes appear as solid, translucent pellet at the bottom of the tube. The supernatant was carefully decanted, and the pellet was rinsed with buffer containing 25 mM KCl, 5 mM MgCl2, 50 mM HEPES-KOH (pH 7.4), and 100 µg/ml cycloheximide to remove residual proteins adhering to it. The pellet containing ribosomes was then resuspended in the same buffer.    To separate polysomes from monosomes, we performed density gradient ultracentrifugation. 10-50% sucrose gradient was prepared as described above. The ribosome-containing sample (from the previous step) was carefully loaded on top of the gradient. The tubes were centrifuged at 29,000 rpm (144208 RCFmax; SW 41 Ti rotor; LE-70 ultracentrifuge, Beckman Coulter) for 4 hours at 4 °C. After centrifugation, 300 μl fractions were collected and absorbance at 260 nm was measured using BioPhotometer D20 (Eppendorf). A total of 30 fractions were collected. Fractions 11-17 constitute the monosomes and fractions 21-27 constitute the polysomes. The fractions were pooled and concentrated using Vivaspin 500 100K MWCO PES concentrator (Sartorius). The tubes were centrifuged at 5000 rpm at 4 °C until the desired concentrated volume is achieved (~100-150 μl). OD at 260 nm was measured for the concentrated samples.     “Negative staining and Transmission electron microscopy”: 3 μl of the concentrated monosome and polysome fractions (derived from sucrose gradient as described above) was applied on a formvar/carbon (10 nm formvar and 1 nm carbon) coated copper grids (400 mesh), (Electron Microscopy Sciences, FCF400-Cu) and incubated for 1 minute. Excess sample was removed using blotting paper. 3 μl of 2% uranyl acetate (Electron Microscopy Sciences, 22400) was added to the grid and incubated for 30 seconds. The excess stain was removed using blotting paper. The grids were then visualized using transmission electron microscope (Tecnai T12, FEI).