80S initiation complexes were formed as previously described (Eyler and Green, 2011 (link)) with minor differences. Briefly, 3 pmol of 35S-Met-tRNAiMet was mixed with 25 pmol of eIF2 and 1 mM GTP in 1X Buffer E (20 mM Tris pH 7.5, 100 mM KOAc pH 7.6, 2.5 mM Mg(OAc)2, 0.25 mM Spermidine, and 2 mM DTT) for 10 minutes at 26°C. Next a mixture containing 25 pmol 40S subunits, 200 pmol T7-synthesized mRNA template, 150 pmol eIF1, and 150 pmol eIF1A in 1X Buffer E was added for 5 minutes. To form the 80S complex, a mixture containing 25 pmol 60S subunits, 125 pmol eIF5, 125 pmol eIF5b, and 1 mM GTP in 1X Buffer E was added for 1 minute. Complexes were then mixed 1:1 with buffer E containing 17.5 mM Mg(OAc)2 to yield a final magnesium concentration of 10 mM. Ribosomes were then pelleted through a 600 μl sucrose cushion containing 1.1 M sucrose in buffer E with 10 mM Mg(OAc)2 using a MLA-130 rotor (Beckmann) at 75,000 rpm for 1 hour at 4°C. After pelleting, ribosomes were resuspended in 25 μl of 1X Buffer E containing 10 mM Mg(OAc)2 and stored at -80°C.
Mla 130 rotor
Manufactured by Beckman Coulter
Sourced in United States
The MLA-130 rotor is a high-performance centrifuge rotor designed for use with Beckman Coulter's laboratory equipment. It is capable of reaching maximum speeds of up to 130,000 RPM, enabling efficient separation and purification of small sample volumes.
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Lab products found in correlation
Optima max xp, by Beckman Coulter (3 mentions)
Exosome depleted fbs, by System Biosciences (2 mentions)
Victor 3 multi label microplate reader, by PerkinElmer (1 mentions)
Amplex red glucose assay kit, by Thermo Fisher Scientific (1 mentions)
Optima ultracentrifuge, by Beckman Coulter (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Exoquick serum exosome precipitation solution, by System Biosciences (1 mentions)
Exoeasy kit, by Qiagen (1 mentions)
Polyu, by Merck Group (1 mentions)
Pabg1 100, by Proteintech (1 mentions)
Sw41 rotor, by Beckman Coulter (1 mentions)
Centrifuge tube, by Beckman Coulter (1 mentions)
Nbd pe, by Avanti Polar Lipids (1 mentions)
Cell disruption bomb, by Parr (1 mentions)
15 protocols using mla 130 rotor
1
Reconstitution of 80S Initiation Complexes
2
Glucose and Insulin Quantification in Mouse Cortex
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Glucose was measured using the Amplex red glucose assay kit (Invitrogen) following the manufacturer’s instructions. Mouse hemi-cortices were lysed in 20 mM Tris HCl pH 7.5, 150 mM NaCl 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, and phosphatases and proteases inhibitors by trituration using a glass-glass dounce tissue homogenizer on ice. Lysates were spun at 30,000 x g using a Beckman Optima Ultra Centrifuge and the MLA 130 Rotor for 30 min at 4 °C. The supernatant was collected for glucose measurement with Amplex red. The fluorescence values were read at 545 nm excitation and 590 nm emission wavelengths using a Victor 3 Multi-label Microplate Reader (Perkin Elmer).
Cortical insulin content was measured using a specific sandwich ELISA for mouse insulin (Millipore) following the manufacturer’s instructions.
Cortical insulin content was measured using a specific sandwich ELISA for mouse insulin (Millipore) following the manufacturer’s instructions.
3
Brain Membrane Protein Extraction Protocol
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Brain membrane proteins were prepared for use in Western blot analysis and co-immunoprecipitation as described57 (link). Briefly, animals were anaesthetized with isoflurane, decapitated, brains were dissected with brainstem and cerebellum removed, then rapidly frozen in 2-methylbutane and stored at −80 °C until use. Frozen brains were placed into ice-cold Tris-EGTA (50 mM Tris, 10 mM EGTA, pH 8.0) containing Complete protease inhibitors at 2X the recommended concentration (Roche Diagnostics). From this point on, samples were kept on ice. Tissue was homogenized using a polytron homogenizer followed by 20 strokes using a glass Dounce homogenizer. Homogenates were centrifuged at 2500 × g for 10 min at 4 °C, and the supernatant was then centrifuged at 377,000 × g in a Beckman-Coulter MLA-130 rotor for 15 minutes at 4 °C. Membranes were resuspended in Tris-EGTA plus protease inhibitors, sonicated, and aliquots stored at −80 °C until use.
4
Preparation of Crude 80S Ribosome Solution from HeLa Cells
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HeLa cell extract was prepared as described previously (Khatter et al., 2014 (link)). A frozen HeLa cell pellet was thawed and suspended with an equal volume of lysis buffer (20mM HEPES pH 7.5, 10mM KOAc, 1.8mM Mg(OAc)2 and 1mM DTT). After incubation on ice for 20 min, the cells were lysed with a Dounce homogenizer 150 times, followed by centrifugation twice at 1,200 x g for 5 min. Supernatant was then aliquoted, flash-frozen with liquid nitrogen and stored at −80°C.
Crude 80S solutions were prepared from HeLa cell lysate using a previously described protocol (Jan et al., 2014 (link)), with the following modifications. Lysate was loaded on 50% sucrose cushion prepared in Buffer A (20mM Tris pH 7.5, 2mM Mg(OAc)2, 150mM KCl), with the addition of 1mM dithiothreitol (DTT). Sucrose cushions were then centrifuged at 100,000 rpm using an MLA-130 rotor (Beckman Coulter) for 60 min at 4°C. The resulting pellet was resuspended in cold Buffer A to homogeneity. Resuspended ribosome pellet was then centrifuged at 21130 x g for 10 min at 4°C to remove any remaining cell debris. Supernatant was then aliquoted, flash-frozen with liquid nitrogen and stored at −80°C. 80S concentration was calculated as described previously (Algire et al., 2002 (link); Jan et al., 2014 (link)).
Crude 80S solutions were prepared from HeLa cell lysate using a previously described protocol (Jan et al., 2014 (link)), with the following modifications. Lysate was loaded on 50% sucrose cushion prepared in Buffer A (20mM Tris pH 7.5, 2mM Mg(OAc)2, 150mM KCl), with the addition of 1mM dithiothreitol (DTT). Sucrose cushions were then centrifuged at 100,000 rpm using an MLA-130 rotor (Beckman Coulter) for 60 min at 4°C. The resulting pellet was resuspended in cold Buffer A to homogeneity. Resuspended ribosome pellet was then centrifuged at 21130 x g for 10 min at 4°C to remove any remaining cell debris. Supernatant was then aliquoted, flash-frozen with liquid nitrogen and stored at −80°C. 80S concentration was calculated as described previously (Algire et al., 2002 (link); Jan et al., 2014 (link)).
5
Purification of MinD, eGFP-MinD, and MinE Proteins
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MinD, eGFP-MinD as well as WT MinE and MinE mutants were expressed and purified as His-tagged fusions as described previously [11 (link), 21 (link)]. After purification, any precipitates were removed by centrifugation in an MLA-130 rotor (Beckman Coulter, Brea, CA, USA) at 50,000 rpm for 30 min at 4°C. Protein quantification was performed by Bradford assay (Bio-Rad Protein Assay, Bio-Rad Laboratories Inc., Hercules, CA, USA). All protein samples were frozen in storage buffer (50 mM Hepes pH 7.25, 150 mM KCl, 10% glycerol, 0.1 mM EDTA, 0.2 mM TCEP; and for MinD 0.2 mM ADP) and stored at -80°C.
6
Exosome Isolation from Serum Using Differential Centrifugation and Kits
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Exosomes were isolated from serum samples using differential centrifugation and two commercial kits: ExoQuick Serum Exosome Precipitation Solution (System Biosciences, Palo Alto, CA, USA) and exoEasy kit (Qiagen GmbH, Hilden, Germany). Serum samples were centrifuged at 2,000×g for 10 min and 10,000×g for 30 min at 4 °C to thoroughly remove cellular debris. Next, supernatants were filtered through a 0.2 μm pore size filter to exclude particles > 200 nm in diameter. For exosome isolation by differential ultracentrifugation, the filtered supernatant was collected into ultracentrifuge tubes and centrifuged in an Optima MAX-XP ultracentrifuge with an MLA-130 rotor (Beckman Coulter, Jersey City, NJ, USA) at 100,000 g for 1 h at 4 °C. The pellets were washed with PBS, ultracentrifuged again, and resuspended in PBS. For exosome isolation by ExoQuick and exoEasy, the two commercial kits were used according to the manufacturers’ instructions. Exosome preparations were conserved at − 80 °C for later use.
7
Ribosome Binding Assay for Upf1
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Ribosomal subunits from S. cerevisiae were purified as previously reported (45 (link)), or from Escherichia coli as previously reported (59 (link)). Ribosome pelleting assays were conducted by incubating ribosomal subunits with Upf1 proteins in reactions containing 100 nM 40S and 60S ribosomal subunits, 100 nM Upf1, 1 mM nucleotide, and 0.2 mg/ml polyU (Sigma) in 1X Buffer E (20 mM Tris, pH 7.5, 100 mM KOAc pH 7.5, 2.5 mM Mg(OAc)2, 0.25 mM spermidine and 2 mM DTT). Reactions were pelleted over sucrose cushions containing 1.1 M sucrose in Buffer E using a Beckman MLA-130 rotor at 75 000 rpm for 1 hour at 4°C. Pelleted ribosomes were resuspended in Buffer E and run on SDS-PAGE gels for Western blotting using an anti-MBP antibody (NEB) to detect Upf1.
8
Recombinant Protein Expression and Ribosome Isolation
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HEK293T cells were seeded in 6-well plates as previously described. The following day, cells were transfected with 2 µg of expression plasmids coding for recombinant SFL, SFLS, or SFL mutants carrying a cMYC-tag at the C-terminus. A vector expressing dsRed tagged with a cMYC-epitope and an empty vector served as controls. At 48 h post-transfection, cells were harvested in cold medium and subsequently pelleted via centrifugation for 3 min at 2300 g and washed with ice-cold PBS. Cells were lysed with 250 µL RNC buffer (50 mM HEPES-KOH, pH 7.4, 100 mM KCl; 5 mM MgCl2) supplemented with 100 µg/mL cycloheximide and Triton X-100 at a final concentration of 0.1%. After a 2 h incubation on ice, lysates were centrifuged for 30 min at 13,400 g at 4 °C to remove cell debris. A total of 180 µL of the supernatant was loaded onto 800 µL of a 0.5 M sucrose cushion in RNC buffer and the remaining whole cell lysate served as input control. Samples were centrifuged for 1 h at 100,000 rpm at 4 °C (MLA-130 rotor, Beckman Coulter, Krefeld, Germany), and, subsequently, the supernatant was discarded. The ribosomal pellet was carefully washed with 100 µL RNC buffer, resuspended in 45 µL of RNC buffer, and proteins of interest were detected via immunoblotting.
9
Purification of Organelles from Activated B Cells
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The procedure was performed as described previously33 (link),54 (link). Briefly, activated B cells from Tent5c-GFP knock-in mice were lysed in MTE buffer (270 mM D-mannitol, 10 mM Tris pH 7.4, 0.1 mM EDTA, 1 mM PMSF) by homogenization monitored by microscopy. Extracts were cleared by sequential centrifugation at 700 and 15,000 × g and subsequently were loaded on the top of the discontinuous sucrose gradient (1.3 M, 1.5 M, and 2 M prepared in 10 mM Tris pH 7.6, 0.1 mM EDTA) and ultracentrifuged at 152,000 × g for 70 min in an SW41 rotor (Beckman). The top layer was collected as cytosol fraction. ER fraction was collected as band at the interphase of 1.3 M sucrose layer, diluted with additional MTE buffer and ultracentrifuged at 126,000 × g for 45 min in MLA130 rotor (Beckman), and pellet was collected as purified ER, resuspended in PBS supplemented with proteases inhibitors and subsequently analyzed using western blot and antibodies against TRAPα (Clone EPR5603), SSR3 (Abcam, ab190936), HDLBP (Bethyl, A303-971A), and PERK (Clone C33E10, GRP94 (Clone H-212), CHOP (Clone D46F1), and GFP (ChromoTek, PABG1-100)).
10
Liposome Formation from DNA Nanostructures
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A solution containing 10 nM of DNA nanostructure, 240 nM of Alexa Fluor 647 modified DNA, 480 nM of lipid modified DNA and 0.67% OG in 25 mM HEPES, 10 mM MgCl2 was incubated at 37 °C for 1 h. To form DNA-ring templated liposomes, 15 µL of 25 mM HEPES, 10 mM MgCl2 (pH 7.5), 2 µL of 6.7% OG, 6 µL 4 M KCl and 5 µL of 15 mM DOPC (1% Rhod-PE) were added to 40 µL of 10 nM lipid- and Alexa Fluor 647-labeled DNA rings to reach 60 µL total volume and the solution was shaken for 30 min at room temperature. The solution was diluted with 60 µL of 25 mM HEPES, 10 mM MgCl2 and 0.4 M KCl buffer containing 0.67% OG and dialyzed against 2 L of buffer overnight. The amount of 100 µL of recovered solution was mixed with 200 µL of 30% iodixanol in 1× hydration buffer and placed at the bottom of a centrifuge tube (11 × 34 mm, Beckman Coulter Inc., Brea, CA, USA). Six additional layers of 0 to 15% iodixanol solution in buffer were added to the centrifuge tube. The tube was spun in an MLA-130 rotor (Beckman Coulter Inc. Brea, CA, USA) at 237,000× g and 4 °C for 5 h, then after the fractions (52 μL per fraction) were collected.
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