Whole yeast cell extracts obtained by cryogenic milling on a Ball Mill device (Retsch PM 100) from the same strains used in the affinity purification assays were fractionated after density gradient sedimentation as previously described (41 (link),77 (link)). Briefly, pre-cleared lysates − obtained from Nop53 depleted (−) or non-depleted (+) cells − corresponding to 5 mg of total protein were loaded on the top of a 12 ml linear 10–30% glycerol gradient (20 mM Tris–HCl pH 8.0, 200 mM KCl, 5 mM MgOAc, 5% glycerol, 1 mM PMSF), previously obtained using a Gradient Master device (BioComp), and were centrifuged at 23 000 rpm for 12.5 h at 4°C in a P40ST rotor (Hitachi). Each gradient was fractionated from the top in 24 aliquots (500 μl) using an EconoSystem (Bio-Rad) fitted with a UV monitor plotting the protein/nucleic acid sedimentation profile. All the gradient fractions were flash-frozen in liquid N2 and later precipitated with ice-cold 10% trichloroacetic acid (TCA) before analysis by SDS-PAGE and western blot. The 48 gradient fractions (24 Nop53+ and 24 Nop53–) of each strain were processed and analyzed together. The same amount of Input was loaded in each gel. All the western blot membranes (Nop53+ and Nop53–) of the same strain were scanned together under the same parameters using the Odyssey Imaging System (LI-COR) with near-infrared fluorescence detection.
Gradient master device
Manufactured by BioComp Instruments
Sourced in Canada
The Gradient Master device is a laboratory instrument designed for the creation of linear gradients. It is used to generate gradients of varying concentrations, typically for applications in fields such as chromatography and electrophoresis. The device operates by precisely controlling the mixing of two or more solutions to produce a continuous gradient.
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Lab products found in correlation
Piston gradient fractionator, by BioComp Instruments (5 mentions)
Sw60ti rotor, by Beckman Coulter (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Econosystem, by Bio-Rad (1 mentions)
P40st rotor, by Hitachi (1 mentions)
Tls 55 rotor, by Beckman Coulter (1 mentions)
Optilab rex refractive index detector, by Wyatt Technology (1 mentions)
Dawn heleos mals detector, by Wyatt Technology (1 mentions)
Tskgel supersw2000 column, by Tosoh (1 mentions)
Astra software, by Wyatt Technology (1 mentions)
Xl 90 ultracentrifuge, by Beckman Coulter (1 mentions)
10 protocols using gradient master device
1
Yeast Cell Extract Fractionation
2
Glycerol Gradient Fixation of PaFS Complex
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Glycerol gradient fixation (GraFix) was performed as described by Kastner et al.28 (link). In brief, PaFS (61 μM) was incubated on ice for 30 min in EM buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM TCEP] before ultracentrifugation. Samples were sedimented for 10 h at 4°C, 45,000 × g in a 10–40% glycerol gradient of EM buffer using a Beckman SW 60 Ti rotor. Glycerol gradients were prepared using a Gradient Master device (BioComp Instruments). When crosslinking was used for EM analysis, 0.125% (v/v) glutaraldehyde was added to the 40% glycerol solution before gradient preparation. Samples were then fractionated with a Piston Gradient Fractionator (BioComp Instruments). Crosslinking reactions were quenched by the addition of glycine-HCl buffer (pH 7.5) to a final concentration of 40 mM. Finally, the crosslinked PaFS complex was dialyzed into EM buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM TCEP] for 60 minutes to remove glycerol.
3
Purification of Modified N Proteins
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Bacterial extracts from E. coli cells expressing 6×His-tagged N, Nm4m1, Nm6m1, Nm4m2, or Nm6m2 fusion proteins were loaded on top of a 2.2-ml continuous glycerol gradient of 15 to 40% (wt/vol) in lysis buffer, prepared using a Gradient Master device (BioComp, Canada). After centrifugation at 135,000 × g for 75 min at 4°C in a TLS55 rotor (Beckman Coulter, Brea, CA), 11 fractions of 200 μl were collected from the top to the bottom of the gradient. After precipitation with the addition of 20 μl of trichloroacetic acid (final concentration, 10%), proteins were resolved by SDS-PAGE and analyzed by Western blotting using monoclonal antibodies against the MHV N protein.
4
Sucrose Density Gradient Fractionation
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Lysates obtained as described above were loaded on a 15–40% linear sucrose density gradient. Two 15 and 40% sucrose buffers were prepared in 10 mM Tris pH 7.4; 10 mM MgCl2; 250 mM KCl; 25 mM EGTA; 1 mM DTT. To obtain a linear gradient, we used the Gradient Master device (BioComp) equipped with tubes adapted for a SW41 rotor. We used 6 ml of each sucrose buffer in each gradient tube. In the ultracentrifuge tube, the light sucrose solution was underlayered with the heavy sucrose solution using a cannula attached to a 10 ml syringe. The introduction of air bubbles or mixing of the two solutions should be carefully avoided during this step. The ‘SW41 Short Gradient’ program with the corresponding range of sucrose percentage was run following the manufacturer's instructions. This program lasts a few minutes (2 min 21 s for a 15–40% gradient) and produces up to six reproducible linear gradients in one run. The gradients should be handled with care to avoid disturbance and stored at 4°C for at least 1 h. The percentage of sucrose used for linear gradients can be adjusted to optimize the separation of polysomal fractions according to the biological sample or experiment.
5
Sucrose Gradient Fractionation of Protein Complexes
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Flag-peptide eluates (max. 500μl) of tandem affinity purifications were further resolved by sucrose gradient ultracentrifugation. Therefore, 95% of the eluted yeast samples were loaded onto a linear 15%–40% (w/v) sucrose gradient containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 0.001% v/v NP-40, and 1 mM DTT and centrifuged for 16 hours at 129,300 xg and 4°C. Sucrose gradients were made using the Gradient Master device (BioComp Instruments). Fractions from the sucrose gradient were harvested by using the Foxy Junior® fraction collector and pooled to final fractions of ~1.2 ml. The fractions were TCA precipitated and analyzed SDS-PAGE and Coomassie staining.
6
Glycerol Gradient Centrifugation for Protein Analysis
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Glycerol gradient centrifugation with (GraFix) or without fixation was performed similarly to Kastner et al. (52 (link)). Briefly, proteins were sedimented at 40,000 RPM for ∼16 h in a 5 to 30% glycerol gradient at 4 °C using a Beckman SW 60 Ti rotor. Gradients were prepared in 20 mM Hepes (pH 7.5), 0.2 M NaCl, 5 mM MgCl2, and 1 mM EGTA using a Gradient Master device (BioComp Instruments). For experiments with crosslinking, 0.125% (v/v) glutaraldehyde was added to the 30% glycerol solution before preparing the gradient. Crosslinking was quenched by the addition of 40 mM glycine–HCl (pH 7.5). A Piston Gradient Fractionator (BioComp Instruments) was used for fractionation.
Glycerol in samples was removed by quick dialysis, followed by centrifugal concentration. For mass determination, samples were injected into a TSKgel SuperSW2000 column (Tosoh Corporation) before entering a DAWN HELEOS MALS detector and an Optilab rEX refractive index detector (Wyatt Technology Corporation). The Astra software (Wyatt Technology Corporation) was used to calculate molecular masses.
Glycerol in samples was removed by quick dialysis, followed by centrifugal concentration. For mass determination, samples were injected into a TSKgel SuperSW2000 column (Tosoh Corporation) before entering a DAWN HELEOS MALS detector and an Optilab rEX refractive index detector (Wyatt Technology Corporation). The Astra software (Wyatt Technology Corporation) was used to calculate molecular masses.
7
Reconstitution of DNA Damage Repair Complex
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To prepare samples for competition assays, 120 pmol of Rad4–Rad23, 120 pmol of Rad33, and 120 pmol of −24/+24 AAF were mixed and dialyzed to assembly buffer (20 mM HEPES pH 7.6, 300 mM potassium acetate, 2 mM magnesium acetate, 5% (v/v) glycerol, 5 mM DTT) for 4 h. Dialyzed Rad4–Rad23–Rad33/AAF was then added to 0.83 nmol of coreTFIIH with 5 mM ATP or 2 mM AMP-PNP 2 mM magnesium acetate and 120 pmol of Rad14 or Rad2. Reactions were incubated for 60 min. TFIIH/Rad4–Rad23–Rad33/AAF + /− Rad14/Rad2 were then sedimented at 52,000 rpm for 6 h and 35 min in a 10 to 40% glycerol gradient (20 mM HEPES pH 7.6, 150 mM potassium acetate, 2 mM magnesium acetate, 4 mM DTT, 2 mM ATP or 1 mM AMP-PNP) at 4 °C using a Beckman SW 60 Ti rotor. Glycerol gradients were prepared using a Gradient Master device (BioComp Instruments). Samples were then fractionated with a Piston Gradient Fractionator (BioComp Instruments). Protein distribution was analyzed by TCA precipitation of fractions followed by SDS-PAGE analysis. Gel images were scanned and then densitometry analysis was performed in ImageJ90 (link) and data were plotted in Prism (GraphPad). Data points and raw gels provided in the Source Data file.
8
Stabilization and Isolation of Protein Complexes
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Complexes were stabilised and isolated using the GraFix technique28 (link). Complexes were formed in the same molar conditions and incubated in the same buffer as for gel filtration analysis. After incubation (60 min, room temperature), samples were deposited on a 10–30% glycerol gradient in incubation buffer (100 mM KCl, 5 mM MgCl2, 20 mM Hepes pH 7.4). Continuous gradients were formed using a Gradient Master device (BioComp). The heavy solution contained 0.15% glutaraldehyde; as a control, a glutaraldehyde-free gradient was run in parallel. Samples were ultracentrifuged (132,000 xg, 16 hr, 4 °C) using an SW55 Ti rotor in a Beckman XL-90 ultracentrifuge. Fractions were collected manually from the top of the tube and analysed by SDS-PAGE in 10% or 6% polyacrylamide gels for controls and crosslinked samples, respectively. Gels were silver-stained using standard protocols and positive fractions evaluated by EM.
9
Arp2/3 Complex Sedimentation Analysis
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Glycerol gradient fixation was performed similarly to Kastner et al. (39 (link)). Arp2/3 complex (5 μM) was mixed with rabbit skeletal α-actin (15 μM) and GCA (20 μM). The same molar ratio was used to assemble Arp2/3 complex with MBP-GCA and actin. Proteins were incubated on ice for 30 min before ultracentrifugation. Samples in Arp buffer supplemented with 0.4 mM MgCl2 and 0.2 mM adenosine 5′-triphosphate (ATP) were sedimented at 40,000 rpm for ~16 hours in a 5 to 30% glycerol gradient at 4°C using a Beckman SW 60 Ti rotor. Glycerol gradients were prepared using a Gradient Master device (BioComp Instruments). When cross-linking was used for EM analysis, 0.125% (v/v) glutaraldehyde was added to the 30% glycerol solution before gradient preparation. Samples were then fractionated with a Piston Gradient Fractionator (BioComp Instruments). Cross-linking reactions were quenched by the addition of glycine-HCl buffer (pH 7.5) to a final concentration of 40 mM. Before EM analysis, Arp2/3 complex was dialyzed into Arp buffer supplemented with 0.4 mM MgCl2 and 0.2 mM ATP to remove glycerol.
10
Reconstitution of TFIIH-Rad4-Rad23 Complex
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To prepare samples for competition assays, 120 pmoles of Rad4-Rad23, 120 pmoles of Rad33 and 120 pmoles of -24/+24 AAF were mixed and dialyzed to assembly buffer (20 mM HEPES pH 7.6, 300 mM potassium acetate, 2 mM magnesium acetate, 5% (v/v) glycerol, 5 mM DTT) for 4 hours. Dialyzed Rad4-Rad23-Rad33/AAF was then added to 0.83 nmoles of coreTFIIH with 5 mM ATP or 2 mM AMP-PNP 2 mM magnesium acetate and 120 pmole of Rad14 or Rad2. Reactions were incubated for 60 minutes. TFIIH/Rad4-Rad23-Rad33/AAF +/-Rad14/Rad2 were then sedimented at 52,000 rpm for 6 hours and 35 minutes in a 10 to 40% glycerol gradient (20 mM HEPES pH 7.6, 150 mM potassium acetate, 2 mM magnesium acetate, 4 mM DTT, 2 mM ATP or 1 mM AMP-PNP) at 4°C using a Beckman SW 60 Ti rotor. Glycerol gradients were prepared using a Gradient Master device (BioComp Instruments). Samples were then fractionated with a Piston Gradient Fractionator (BioComp Instruments). Protein distribution was analyzed by TCA precipitation of fractions followed by SDS-PAGE analysis. Gel images were scanned and then densitometry analysis was performed in ImageJ 77 and data were plotted in Prism (GraphPad).
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