Gradient master 108

Manufactured by BioComp Instruments

Sourced in Canada

The Gradient Master 108 is a laboratory instrument designed for creating linear gradients. It utilizes two peristaltic pumps to precisely control the flow and mixing of solutions, allowing for the creation of gradient solutions with user-defined parameters.

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Lab products found in correlation

Piston gradient fractionator, by BioComp Instruments (7 mentions) Sw60 swinging bucket rotor, by Beckman Coulter (2 mentions) Thinwall ultra clear tube, by BioComp Instruments (2 mentions) Gel filtration markers kit, by Merck Group (2 mentions) Optima l 100 xp, by Beckman Coulter (2 mentions) Vacufuge plus, by Eppendorf (2 mentions) Pgfip piston gradient fractionator, by BioComp Instruments (2 mentions) Hiseq 2500, by Illumina (2 mentions) Nanodrop, by Thermo Fisher Scientific (2 mentions) Th 641, by Thermo Fisher Scientific (1 mentions) Foxy r1, by Teledyne (1 mentions) Rnasin, by Merck Group (1 mentions) Uv monitor, by Bio-Rad (1 mentions) Rneasy kit, by Qiagen (1 mentions) T100 thermal cycler, by Bio-Rad (1 mentions) Sw41ti rotor, by Beckman Coulter (1 mentions) Kontes 99 mm filtration apparatus, by Cytiva (1 mentions) 0.45 μm nitrocellulose filter, by Cytiva (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions) Truseq stranded total rna kit, by Illumina (1 mentions)

Spelling variants of this product

Gradient Master (110 citations) Model 108 gradient master (3 citations) BioComp Gradient Master 108 (3 citations)

29 protocols using gradient master 108

Cell Lysis and Fractionation

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30 ODs of cells were harvested in PBS buffer, fresh 0.5 mM DTT and Roche’s complete protease inhibitor. Lysis was performed by bead-beating (Zirconia-Silicon beads). Glycerol gradients were made using Gradient Master 108 from Biocomp. Centrifugation was performed at 100,000 xg for 4 hr at 4°C. 1 mL fractions were collected from the solutions, TCA precipitated and immunoblotted as described above.

Banjade S., Tang S., Shah Y.H, & Emr S.D. (2019). Electrostatic lateral interactions drive ESCRT-III heteropolymer assembly. eLife, 8, e46207.

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Purification of Virus-Like Particles

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VLPs were separated from contaminants according to their buoyant density by sedimentation in CsCl gradients. CsCl solutions were prepared in 50 mM sodium phosphate, pH 7 buffer containing 100 mM sodium chloride at concentrations of 1.2 and 1.4 g/mL. The 1.4 g/mL solution was deposited below the 1.2 g/mL solution in centrifugation tubes compatible with Thermo Fisher swinging bucket rotor TH-641. Using the Biocomp Gradient Master 108 preset setting, a 12−14% (w/w) CsCl gradient was prepared. A concentrated solution of protein (>8 mg/mL 750 μL) was deposited in each centrifugation tube containing the gradient and balanced. The tubes were centrifuged at 38 000 rpm for 2 h. Afterward, a band was formed in the region where the particles were deposited. Fractions were collected from that band and used for further experimentation.

Selivanovitch E., Koliyatt R, & Douglas T. (2018). Chemically Induced Morphogenesis of P22 Virus-like Particles by the Surfactant Sodium Dodecyl Sulfate. Biomacromolecules, 20(1), 389-400.

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RNA-seq Sample Preparation from Organoids

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Organoids were treated with vehicle or 10 nM CR-31 for 1 h. In the final 5 min, 100 μg ml^–1 of Cycloheximide was supplemented into the media. The organoids were then harvested on ice in PBS containing 100 μg ml^–1 Cycloheximide. Cells were pelleted and lysed in 10 mM Tris-HCl (pH 8), 140 mM NaCl, 1.5 mM MgCl₂, 0.25% NP-40, 0.1% Triton X-100, 50 mM DTT, 150 μg ml^–1 Cycloheximide, and 640 U ml^–1 RNasin (Sigma Cat# 3335399001) for 15 min. Lysates were cleared, and then loaded onto a 10–50% sucrose gradient made using a Biocomp Gradient Master 108 and centrifuged for 2 h and 15 min at 151,263 × g in a SW41 rotor using a Sorvall Discovery 90SE. The gradients were fractionated on a Teledyne ISCO Foxy R1 apparatus while monitoring the OD₂₅₄.

Chan K., Robert F., Oertlin C., Kapeller-Libermann D., Avizonis D., Gutierrez J., Handly-Santana A., Doubrovin M., Park J., Schoepfer C., Da Silva B., Yao M., Gorton F., Shi J., Thomas C.J., Brown L.E., Porco JA J.r., Pollak M., Larsson O., Pelletier J, & Chio I.I. (2019). eIF4A supports an oncogenic translation program in pancreatic ductal adenocarcinoma. Nature Communications, 10, 5151.

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Vps24 glycerol gradient analysis

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For Vps24 and Vps24 E114K glycerol gradients, vps24∆ was transformed with pCM189 Vps24 or pCM189 Vps24 E114K. Thirty ODs of cells expressing these constructs were harvested in phosphate saline buffer (PBS). Lysis was performed with PBS buffer, 10% glycerol, 1 mM DTT, Roche protease cocktail, and 0.5% Tween-20. Gradient Master 108 from Biocomp was used to make glycerol gradients of 10–40%. Centrifugation was performed at 100,000 × g for 4 hr at 4°C; 1 mL fractions were collected from the solutions, TCA-precipitated, and immunoblotted.

Banjade S., Shah Y.H., Tang S, & Emr S.D. (2021). Design principles of the ESCRT-III Vps24-Vps2 module. eLife, 10, e67709.

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Sucrose Gradient Fractionation of Liver Lysates

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Using the Gradient Master 108 programmable gradient pourer (Biocomp), 17.5 to 50

%

sucrose gradients were generated in gradient buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM

{M g C l}_{2}

, 1 mM DTT, and 100

μ

g/mL Cycloheximide). Liver lysates from AL ZT18 and FA ZT18 containing same amount of RNA were then loaded onto sucrose gradients and centrifuged for 3.5 h at 28,000 rpm in an SW40Ti rotor in a Beckman L7 ultracentrifuge (Beckman Coulter). After centrifugation, gradients were fractionated and measured for RNA content (absorbance at 254 nm) using a Piston Gradient Fractionator (Biocomp) connected to a UV monitor (Bio-Rad).

Gobet C., Weger B.D., Marquis J., Martin E., Neelagandan N., Gachon F, & Naef F. (2020). Robust landscapes of ribosome dwell times and aminoacyl-tRNAs in response to nutrient stress in liver. Proceedings of the National Academy of Sciences of the United States of America, 117(17), 9630-9641.

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Polysome Profiling of Transfected Cells

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A549 cells (transfected with target-specific siRNAs as described above for 72 hours in 10 cm² dishes, seeded at 3.2 × 10⁵ cells/plate) were treated with 100 μg/mL cycloheximide (CHX) for 5 minutes then harvested in hypotonic lysis buffer as similarly described (Chan et al., 2011 (link)). Lysates from equal cell number (8 × 10⁶) were loaded onto high salt (250 mM NaCl, 3.1–30.1%) or low salt (80 mM NaCl, 10-40% (w/v)) sucrose gradients generated using a BioComp Gradient Master 108 and separated by centrifugation (SW41 rotor at 40,000 rpm for 4 hours or 36,000 rpm for 2.15 hours, respectively) using a Beckmann Coulter Optima XE-100 Ultracentrifuge. Samples were fractionated (1 mL fractions) using a Teledyne ISCO Foxy R1 instrument. Absorbance at 260 nm was determined using a Brandel UA-6 UV/Vis detector, and measurement of the area under the peaks in each trace conducted. For conducting analysis of transcripts being translated, RNA was isolated from individual fractions using the Qiagen RNeasy kit as per manufacturers’ instructions. cDNA was then synthesised from samples and analysed using qPCR as described in the relevant sections above.

Hannan K.M., Soo P., Wong M.S., Lee J.K., Hein N., Poh P., Wysoke K.D., Williams T.D., Montellese C., Smith L.K., Al-Obaidi S.J., Núñez-Villacís L., Pavy M., He J.S., Parsons K.M., Loring K.E., Morrison T., Diesch J., Burgio G., Ferreira R., Feng Z.P., Gould C.M., Madhamshettiwar P.B., Flygare J., Gonda T.J., Simpson K.J., Kutay U., Pearson R.B., Engel C., Watkins N.J., Hannan R.D, & George A.J. (2022). Nuclear stabilization of p53 requires a functional nucleolar surveillance pathway. Cell Reports, 41(5), 111571.

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Competitive Regulation of Pol II by PHF3 and TFIIS

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Pol II was phosphorylated with DYRK1A kinase as described above. A nucleic acid scaffold for transcribing Pol II was assembled by mixing equimolar amounts of DNA (EC-phf3-template) and RNA (RNA50) in a BioRad T100 Thermal Cycler heated to 95 °C and cooled in 0.1 °C/s increments until 4 °C was reached. For sucrose gradient ultracentrifugation, the Pol II-EC was assembled by incubating 60 pmol Pol II with a 2-fold molar excess of DNA/RNA for 10 min on ice, followed by 10 min at 30 °C, and another 10 min at 30 °C after adding a 4-fold molar excess of non-template DNA (EC-phf3-nontemplate) to generate a transcription bubble. A 4-fold molar excess of PHF3 or TFIIS^M/TFIIS^M+TFIIF was incubated with Pol II-EC for 20 min at 25 °C, followed by addition of the 4-fold molar excess of the competitor (TFIIS^M/TFIIS^M+TFIIF or PHF3 respectively) for 20 min at 25 °C. 10–30% sucrose gradients were prepared using a gradient mixer (Gradient Master 108; BioComp Instruments). Pol II complexes were applied on top of the gradient followed by ultracentrifugation 105169 g in a SW60 swinging bucket rotor (Beckman Coulter) for 16 h at 4 °C. 80 µl fractions were collected carefully from top to the bottom of the tube and analyzed by Western blotting.

Appel L.M., Franke V., Bruno M., Grishkovskaya I., Kasiliauskaite A., Kaufmann T., Schoeberl U.E., Puchinger M.G., Kostrhon S., Ebenwaldner C., Sebesta M., Beltzung E., Mechtler K., Lin G., Vlasova A., Leeb M., Pavri R., Stark A., Akalin A., Stefl R., Bernecky C., Djinovic-Carugo K, & Slade D. (2021). PHF3 regulates neuronal gene expression through the Pol II CTD reader domain SPOC. Nature Communications, 12, 6078.

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Sucrose Gradient Fractionation of WCE Complexes

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Twelve A₂₆₀ units of WCE were separated by high velocity sedimentation through a 7–30% sucrose gradient containing a top-to-bottom increasing concentration of formaldehyde at 22 200 rpm for 17 h using the Beckman Coulter SW41Ti rotor. Such a gradient was prepared by mixing 30% sucrose in buffer B (10 mM HEPES [pH 7.5], 62.5 mM KCl, 2.5 mM MgCl2, 1 mM DTT) containing 0,05% formaldehyde with 7% sucrose in buffer B lacking formaldehyde using the Gradient Master 108 (Biocomp Instruments). Fractions of 600 μl were collected and precipitated with 100% ethanol overnight at –20°C. After a single washing step with 100% ethanol, the pellet was dried and dissolved in 1× standard loading buffer, boiled to reverse cross-linking, and analyzed by SDS-PAGE followed by western blotting. Catalog numbers for all antibodies used in this study are listed in Supplementary Table S2.

Herrmannová A., Prilepskaja T., Wagner S., Šikrová D., Zeman J., Poncová K, & Valášek L.S. (2019). Adapted formaldehyde gradient cross-linking protocol implicates human eIF3d and eIF3c, k and l subunits in the 43S and 48S pre-initiation complex assembly, respectively. Nucleic Acids Research, 48(4), 1969-1984.

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Subcellular fractionation of bacterial lysates

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Cells were cultured at 37 °C in 500 mL of LB (and antibiotics where appropriate) to OD₆₀₀ = 0.5. For samples with antibiotic treatments, cells were cultured to OD₆₀₀ = 0.45, then treated for 5 min by antibiotics at the concentrations indicated in the figures. Cells were then harvested by filtration using a Kontes 99 mm filtration apparatus with a 0.45 μm nitrocellulose filter (Whatman), and flash frozen in liquid nitrogen. Cells were lysed in lysis buffer (20 mM Tris pH 8.0, 10 mM MgCl₂, 100 mM NH₄Cl, 5 mM CaCl₂, 100 U/mL DNase I, and 1 mM chloramphenicol) using a Spex 6870 freezer mill with 5 cycles of 1 min grinding at 5 Hz and 1 min cooling. Lysates were centrifuged at 20,000 × g for 30 min at 4 °C to pellet cell debris. 10–54% sucrose density gradients were prepared using the Gradient Master 108 (Biocomp) with gradient buffer (20 mM Tris pH 8.0, 10 mM MgCl₂, 100 mM NH₄Cl, and 2 mM DTT). 5–40 AU of E. coli lysate was loaded on top of sucrose gradient and centrifuged in a SW41 rotor at 35,000 rpm for 2.5 h at 4 °C. Fractionation was performed on a Piston Gradient Fractionator (Biocomp). To process each fraction for western blots, proteins were precipitated in 10% trichloroacetic acid (TCA) and the pellets were washed twice by ice-cold acetone, vacuum-dried briefly, resuspended in 5x loading dye, and neutralized with Tris-HCl pH 7.5.

Saito K., Kratzat H., Campbell A., Buschauer R., Burroughs A.M., Berninghausen O., Aravind L., Green R., Beckmann R, & Buskirk A.R. (2022). Ribosome collisions induce mRNA cleavage and ribosome rescue in bacteria. Nature, 603(7901), 503-508.

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Sucrose Gradient Fractionation Protocol

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A 5 ml linear sucrose velocity gradient was created as described by Zhang et al., (2010) (68). The sucrose solution contained 20 mm HEPES-KOH pH 7.5, 2 mm EDTA, 1 mm DTT, 1 mm PMSF, protease inhibitors. A linear 22 to 52% sucrose gradient was formed using a gradient master 108 (BioComp, Fredericton, NB, Canada). Two hundred microliters of the washed P200 pellet was suspended (10 mm HEPES/KOH pH 7.2, 150 mm NaCl, 1 mm EDTA, 10% glycerol, 1% protease inhibitors, 1 mm PMSF) and layered on top of the sucrose gradient. The gradient was centrifuged for 18 h at 100,000 × g, 4 °C in a MLS 50 swinging buck rotor. Twenty-five 200 μl fractions were analyzed.

McBride Z., Chen D., Reick C., Xie J, & Szymanski D.B. (2017). Global Analysis of Membrane-associated Protein Oligomerization Using Protein Correlation Profiling. Molecular & Cellular Proteomics : MCP, 16(11), 1972-1989.

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