Ua 6 uv vis detector, by Teledyne - Product details

1

Ribosome Fractionation from HEK293T Cells

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For ribosome fractionation, cytoplasmic extracts from HEK293T cells were prepared as already described^{13 (link)}. For each sample 1 mg of extract was layered on a 10–50% sucrose gradient containing 20 mM Tris pH 7.6; 80 mM NaCl; 5 mM MgCl₂; 1 mM DTT. The gradients were run in an SW41 Beckman rotor at 220,672 g for 140 min at 4 °C. Following centrifugation gradients were fractionated. Acquisition of the profiles was obtained using the UA6 UV/VIS detector from ISCO.

Tan S., Kermasson L., Hilcenko C., Kargas V., Traynor D., Boukerrou A.Z., Escudero-Urquijo N., Faille A., Bertrand A., Rossmann M., Goyenechea B., Jin L., Moreil J., Alibeu O., Beaupain B., Bôle-Feysot C., Fumagalli S., Kaltenbach S., Martignoles J.A., Masson C., Nitschké P., Parisot M., Pouliet A., Radford-Weiss I., Tores F., de Villartay J.P., Zarhrate M., Koh A.L., Phua K.B., Reversade B., Bond P.J., Bellanné-Chantelot C., Callebaut I., Delhommeau F., Donadieu J., Warren A.J, & Revy P. (2021). Somatic genetic rescue of a germline ribosome assembly defect. Nature Communications, 12, 5044.

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2

Polysome Profiling: Ribosomal Subunit Analysis

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Polysome profiling was performed following the protocols described in (19 ,20 (link)). To obtain the cytoplasmic lysates, cells were treated with cycloheximide (10 μg ml⁻¹) for 3–4 min and then lysed in 300 μl of cold hypotonic lysis buffer (19 ). To remove nuclei, mitochondria and cellular debris, the lysates were centrifuged at 4°C for 5 min at 20 000 g. To separate ribosomal subunits, ribosomes and polysomes from other cytoplasmic molecules, the supernatant was loaded on a 10–40% (w/v) sucrose gradient and centrifuged for 1 h 30 min at 260 000 g at 4°C in a SW41 rotor using a Beckman Optima LE-80 Ultracentrifuge. Twelve 1 ml fractions were collected and the absorbance at 254 nm was monitored with the UA-6 UV/VIS detector (Teledyne Isco). RNA was purified fraction by fraction using the phenol/chloroform extraction method described in (21 (link)). The retro-transcription reaction was performed using the same volume of RNA for all polysomal fractions. The co-sedimentation profile of mRNAs was obtained by calculating the percentage (or fraction) of mRNAs in each fraction by qPCR as described in (22 (link)).

Cella F., Perrino G., Tedeschi F., Viero G., Bosia C., Stan G.B, & Siciliano V. (2023). MIRELLA: a mathematical model explains the effect of microRNA-mediated synthetic genes regulation on intracellular resource allocation. Nucleic Acids Research, 51(7), 3452-3464.

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3

Sucrose Gradient Fractionation of Ribosomes

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All buffers were described in [39 (link)] and all steps performed at 4°C. Briefly, 1 to 2 g of frozen powder from non-treated (22°C), heat treated (37°C for 5 h and 24 h) and recovered (R22°C for 5 h and 24 h) seedlings were suspended in 4 mL of PEB, incubated on ice for 30 min and centrifuged 15 min at 16,000 g to remove debris. Samples were then filtered on 0.2 µM filters (Sarsted), loaded on 8 mL sucrose cushions, and centrifuged in a Beckman SW41 rotor for 18 h at 35,000 rpm. Pellets were re-suspended in 1 mL of RB and kept on ice for 30 min. A short centrifugation (2 min at 5,000 g) was performed to remove the last debris. Finally, 1 mL of supernatant was layered onto 9 mL linear 15–60% sucrose gradients and centrifuged at 38,000 rpm for 6h30. Gradients were fractionated using the Type 11 Optical Unit (Teledyne ISCO) system and a UA-6 UV/VIS Detector (Teledyne ISCO) at 254 nm. Values and ratios for 40S and 60S/80S peaks are available in Table S4.

Darriere T., Jobet E., Zavala D., Escande M.L., Durut N., de Bures A., Blanco-Herrera F., Vidal E.A., Rompais M., Carapito C., Gourbiere S, & Sáez-Vásquez J. (2022). Upon heat stress processing of ribosomal RNA precursors into mature rRNAs is compromised after cleavage at primary P site in Arabidopsis thaliana. RNA Biology, 19(1), 719-734.

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4

Polysome Profiling of Cycloheximide-Treated ES Cells

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ES cells, grown to 80% confluence, were incubated with 1% (vol/vol) of 9 mg/mL cycloheximide (Sigma Aldrich) for 10 minutes at 37°C and then trypsinized. Cells were washed with PBS and lysed at 4°C using a handheld homogenizer (Fisher Scientific) in polysome buffer containing 50 mM Tris-HCl (Fisher Scientific), 240 mM NaCl (Fisher Scientific), 10 mM MgCl₂ (Sigma Aldrich), 5 mM beta-mercaptoethanol (Sigma Aldrich), 250 mM sucrose (Fisher Scientific), 2% Triton X (Sigma Aldrich), 100µg/mL heparin (Alfa Aesar), and 90µg/mL cycloheximide. Lysates were run on 15–55% sucrose gradients containing 25 mM Tris-HCl, 25 mM NaCl and 5 mM MgCl₂. Gradients were centrifuged at 28,000 rpm for 7–8 hours using a Beckman L8-M ultracentrifuge. The gradients were then broken down using an ISCO density gradient fractionator, retriever and UA-6 UV/Vis detector (ISCO).

Singh S.A., Goldberg T.A., Henson A.L., Husain-Krautter S., Nihrane A., Blanc L., Ellis S.R., Lipton J.M, & Liu J.M. (2014). p53-Independent Cell Cycle and Erythroid Differentiation Defects in Murine Embryonic Stem Cells Haploinsufficient for Diamond Blackfan Anemia-Proteins: RPS19 versus RPL5. PLoS ONE, 9(2), e89098.

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5

Purification of Human Ribosomal Subunits

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Preparation of small (40S) and large (60S) human ribosomal subunits was adapted from refs. 45 (link) and 73 . Specific deviations implemented for the purification of polysome fractions from human tissue culture are described here. Cell pellets were resuspended in lysis buffer [20 mM Tris HCl (pH 7.5), 2.5 mM MgCl₂, 10 mM KCl, and 1 mM freshly prepared DTT] with the RNase inhibitor RNase Out (Invitrogen), EDTA-free Halt Protease Inhibitor (Thermo Scientific), and cycloheximide (Sigma) at 100 μg/mL (∼350 µM). The solution was incubated on ice for 10 min before centrifugation in a Microfuge 22R Refrigerated Centrifuge (Beckman Coulter) at 14,000 rpm for 10 min at 4 °C to pellet cell debris. The supernatant was loaded onto precooled 10–50% sucrose density gradients and spun at 35,000 rpm for 3 h at 4 °C in an Optima l-100 XP ultracentrifuge (Beckman Coulter). The gradients were then fractionated using a BR-186-1 Fractionator and a UA-6 UV/Vis detector (Teledyne Isco). Fractions corresponding to polysomes were collected and subsequently pelleted and dissociated into subunits according to ref. 73 . Pelleted subunits were resuspended with storage buffer [30 mM Hepes (pH 7.5), 15 mM MgCl₂, 50 mM NH₄Cl, 2 mM spermidine, 5 mM putrescine, 1 mM DTT, and 6% sucrose] for stable, long-term storage in liquid nitrogen.

Prokhorova I., Altman R.B., Djumagulov M., Shrestha J.P., Urzhumtsev A., Ferguson A., Chang C.W., Yusupov M., Blanchard S.C, & Yusupova G. (2017). Aminoglycoside interactions and impacts on the eukaryotic ribosome. Proceedings of the National Academy of Sciences of the United States of America, 114(51), E10899-E10908.

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6

Purification of NAADP Derivatives

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NAADP derivatives can be separated from NADP, ADP-ribose phosphate, and other nucleotides by anion exchange chromatography on DEAE cellulose. DE-52 cellulose was slurry packed into glass Bio-Rad Econo-Columns (2.5 × 50 cm) to form a resin bed approximately 42 cm high. Water (100 mL) was passed through the column to ensure the pH of the effluent from the column was neutral. The dinucleotide mixture was applied to the column as a dilute solution at pH 7.5, and washed into the bed with 10–20 mL of distilled water. The separation was developed by the application of a linear gradient formed between water (350 mL) and 0.6 M NH₄HCO₃ (350 mL). A flow rate of about 1–2 mL/min is achieved using a peristaltic pump (Teledyne-Isco, TRIS) to produce a slight positive pressure. The separation allows purification of compounds based on charge and NAADP derivatives usually elute into the middle of the gradient, into approximately 320 mM NH₄HCO₃. Fractions were collected using an automatic fraction collector (Teledyne- Isco, Retreiver 500, 200 drops/tube), and the dinucleotides detected by their UV absorbance (ISCO UA-6 UV/Vis Detector). UV absorbing peaks were combined, frozen, and lyophilized. Repeated lyophilization of the sample from water ensures complete removal of volatile NH₄HCO₃, leaving pure, salt free dinucleotide.

Trabbic C.J., Zhang F., Walseth T.F, & Slama J.T. (2015). Nicotinic Acid Adenine Dinucleotide Phosphate Analogs Substituted on the Nicotinic Acid and Adenine Ribosides. Effects on Receptor-Mediated Ca2+ release. Journal of medicinal chemistry, 58(8), 3593-3610.

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7

Polysome Profiling of Human LCLs

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Human lymphoblastoid cell lines (LCLs) from control (CTRL4) and patient (F433, F446) samples were used for polysome profiling. Approximately 2x10⁷ cells were incubated in RPMI medium with 100 µg/ml cycloheximide for 5 minutes at 37°C 5% CO₂, followed by a wash in 1 x PBS containing 100 µg/ml cycloheximide. Cells were collected by centrifugation at 200 x g for 5 minutes at 4°C and re-suspended in 425 µl hypotonic buffer (Hypotonic buffer: (5 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 1.5 mM KCl and 1x protease inhibitor cocktail-EDTA-free). The re-suspended cell mixture was supplemented with 5 μl of 10 mg/ml cycloheximide, 1 μl of 1M DTT and 100 units RNasin. The cell mixture was incubated on ice for 10 minutes followed by 5 second vortex. The mixture was further supplemented with 25 μl of 10% Triton X-100 and 25 μl of 10% sodium deoxycholate, and vortexed again for 5 seconds. To pellet debris the lysates were centrifuged at 16,000 x g for 7 min at 4°C and the supernatant transferred to a new pre-chilled tube. The OD at 260 nm was measured for all lysates and adjusted so that all samples contained the same OD. Lysates were loaded onto sucrose gradients (15-50%) and centrifuged for 2.5 hrs at 40,000 rpm. Polysome profiles were recorded using the Isco UA-6 UV/Vis detector.

Jenkinson E.M., Rodero M.P., Kasher P.R., Uggenti C., Oojageer A., Goosey L.C., Rose Y., Kershaw C.J., Urquhart J.E., Williams S.G., Bhaskar S.S., O’Sullivan J., Baerlocher G.M., Haubitz M., Aubert G., Barañano K.W., Barnicoat A.J., Battini R., Berger A., Blair E.M., Brunstrom-Hernandez J.E., Buckard J.A., Cassiman D.M., Caumes R., Cordelli D.M., De Waele L.M., Fay A.J., Ferreira P., Fletcher N.A., Fryer A.E., Goel H., Hemingway C.A., Henneke M., Hughes I., Jefferson R.J., Kumar R., Lagae L., Landrieu P.G., Lourenço C.M., Malpas T.J., Mehta S.G., Metz I., Naidu S., Õunap K., Panzer A., Prabhakar P., Quaghebeur G., Schiffmann R., Sherr E.H., Sinnathuray K.R., Soh C., Stewart H.S., Stone J., Van Esch H., Van Mol C.E., Vanderver A., Wakeling E.L., Whitney A., Pavitt G.D., Griffiths-Jones S., Rice G.I., Revy P., van der Knaap M.S., Livingston J.H., O’Keefe R.T, & Crow Y.J. (2016). Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts. Nature genetics, 48(10), 1185-1192.

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8

Polysomal Profiling of Cell Lysates

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Polysomal profiling was performed according to previously described protocols (Bernabò et al., 2017 (link)). Briefly, the cells were treated with cycloheximide and then lysed in 300 μL of cold lysis buffer. The lysate was centrifuged at 4°C for 5min at 20.000 g to pellet cell debris. The cytoplasmic lysates loaded on a linear 15%–50% [w/v] sucrose gradient and centrifuged in a SW41Ti rotor (Beckman) for 1 h 40 min at 180.000 g at 4°C in a Beckman Optima Optima XPN-100 Ultracentrifuge. Fractions of 1 mL of volume, were then collected monitoring the absorbance at 254 nm with the UA-6 UV/VIS detector (Teledyne Isco).

Tebaldi T., Zuccotti P., Peroni D., Köhn M., Gasperini L., Potrich V., Bonazza V., Dudnakova T., Rossi A., Sanguinetti G., Conti L., Macchi P., D’Agostino V., Viero G., Tollervey D., Hüttelmaier S, & Quattrone A. (2018). HuD Is a Neural Translation Enhancer Acting on mTORC1-Responsive Genes and Counteracted by the Y3 Small Non-coding RNA. Molecular Cell, 71(2), 256-270.e10.

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9

Polysome Profiling of Human LCLs

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Human lymphoblastoid cell lines (LCLs) from control (CTRL4) and patient (F433, F446) samples were used for polysome profiling. Approximately 2x10⁷ cells were incubated in RPMI medium with 100 µg/ml cycloheximide for 5 minutes at 37°C 5% CO₂, followed by a wash in 1 x PBS containing 100 µg/ml cycloheximide. Cells were collected by centrifugation at 200 x g for 5 minutes at 4°C and re-suspended in 425 µl hypotonic buffer (Hypotonic buffer: (5 mM Tris-HCl (pH 7.5), 2.5 mM MgCl₂, 1.5 mM KCl and 1x protease inhibitor cocktail-EDTA-free). The re-suspended cell mixture was supplemented with 5 μl of 10 mg/ml cycloheximide, 1 μl of 1M DTT and 100 units RNasin. The cell mixture was incubated on ice for 10 minutes followed by 5 second vortex. The mixture was further supplemented with 25 μl of 10% Triton X-100 and 25 μl of 10% sodium deoxycholate, and vortexed again for 5 seconds. To pellet debris the lysates were centrifuged at 16,000 x g for 7 min at 4°C and the supernatant transferred to a new pre-chilled tube. The OD at 260 nm was measured for all lysates and adjusted so that all samples contained the same OD. Lysates were loaded onto sucrose gradients (15-50%) and centrifuged for 2.5 hrs at 40,000 rpm. Polysome profiles were recorded using the Isco UA-6 UV/Vis detector.

Jenkinson E.M., Rodero M.P., Kasher P.R., Uggenti C., Oojageer A., Goosey L.C., Rose Y., Kershaw C.J., Urquhart J.E., Williams S.G., Bhaskar S.S., O’Sullivan J., Baerlocher G.M., Haubitz M., Aubert G., Barañano K.W., Barnicoat A.J., Battini R., Berger A., Blair E.M., Brunstrom-Hernandez J.E., Buckard J.A., Cassiman D.M., Caumes R., Cordelli D.M., De Waele L.M., Fay A.J., Ferreira P., Fletcher N.A., Fryer A.E., Goel H., Hemingway C.A., Henneke M., Hughes I., Jefferson R.J., Kumar R., Lagae L., Landrieu P.G., Lourenço C.M., Malpas T.J., Mehta S.G., Metz I., Naidu S., Õunap K., Panzer A., Prabhakar P., Quaghebeur G., Schiffmann R., Sherr E.H., Sinnathuray K.R., Soh C., Stewart H.S., Stone J., Van Esch H., Van Mol C.E., Vanderver A., Wakeling E.L., Whitney A., Pavitt G.D., Griffiths-Jones S., Rice G.I., Revy P., van der Knaap M.S., Livingston J.H., O’Keefe R.T, & Crow Y.J. (2016). Mutations in SNORD118 cause the cerebral microangiopathy leukoencephalopathy with calcifications and cysts. Nature genetics, 48(10), 1185-1192.

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10

Polysomal Profiling of Cell Lysates

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Polysomal profiling was performed according to previously described protocols (Bernabò et al, 2017 (link)). Briefly, cells were seeded in 10‐cm dishes, treated with CHX (10 µg/ml) for 4 min and then lysed in 300 µl of cold hypotonic lysis buffer [10 mM NaCl, 10 mM MgCl₂•6H₂O, 10 mM Tris–HCl, pH 7.5, 1% Triton X‐100, 0.2 U/μl Ribolock RNase inhibitor (Thermo Scientific), 0.0005 U/μl DNaseI (Thermo Scientific), CHX 10 μg/ml and 1 mM dithio‐threitol, 1% sodium deoxycholate]. The lysate was centrifuged at 4°C for 5 min at 1,620 × g to pellet cell debris. The cytoplasmic lysates loaded on a linear 10–40% [w/v] sucrose gradient and centrifuged in a SW41Ti rotor (Beckman) for 1 h 30 min at 187,813 × g at 4°C in a Beckman Optima XPN‐100 Ultracentrifuge. Fractions of 1 ml of volume were then collected monitoring the absorbance at 254 nm with the UA‐6 UV/VIS detector (Teledyne Isco).

Licata N.V., Cristofani R., Salomonsson S., Wilson K.M., Kempthorne L., Vaizoglu D., D’Agostino V.G., Pollini D., Loffredo R., Pancher M., Adami V., Bellosta P., Ratti A., Viero G., Quattrone A., Isaacs A.M., Poletti A, & Provenzani A. (2021). C9orf72 ALS/FTD dipeptide repeat protein levels are reduced by small molecules that inhibit PKA or enhance protein degradation. The EMBO Journal, 41(1), e105026.

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Ua 6 uv vis detector

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14 protocols using ua 6 uv vis detector

Ribosome Fractionation from HEK293T Cells

Polysome Profiling: Ribosomal Subunit Analysis

Sucrose Gradient Fractionation of Ribosomes

Polysome Profiling of Cycloheximide-Treated ES Cells

Purification of Human Ribosomal Subunits

Purification of NAADP Derivatives

Polysome Profiling of Human LCLs

Polysomal Profiling of Cell Lysates

Polysome Profiling of Human LCLs

Polysomal Profiling of Cell Lysates

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