RNA samples (1–2 mg/ml) were exchanged extensively into standard buffer (described above) with 0–50 mM putrescine•(HCl)2 as indicated. The samples were heated to 65°C for 5 min and incubated at room temperature for at least 30 min. Samples were then passed through a 0.1 μm filter (Millipore) prior to beam exposure. SAXS measurements were performed at beamline 12-ID at the Advanced Photon Source, Argonne National Laboratory. The beam energy was set to 12 keV with an exposure time of 0.5 s. Samples were moved through an X-ray flow cell to minimize radiation damage. The ambient temperature was ∼25°C. Thirty shots were collected for each sample condition in order to obtain good statistics. Radii of gyration (Rgs) were determined from the Guinier fit to averaged data; P(r) plots were generated using GNOM after finding approximate DMax values in AUTOGNOM (23 ). Envelopes were generated for the N and Ex-state using DAMMIF. The results of the simulated annealing procedure were uninterruptable for the Ex-state mutant in normal P1 mode, and the simulation was therefore rerun with P2 symmetry imposed.
0.1 μm filter
Manufactured by Merck Group
Sourced in United States
The 0.1 μm filter is a laboratory equipment designed to remove particles and contaminants from liquids or gases. It has a pore size of 0.1 micrometers, which allows the passage of smaller molecules while effectively trapping larger particles and impurities.
Automatically generated - may contain errors
Lab products found in correlation
Cf200h cu, by Electron Microscopy Sciences (2 mentions)
Tecnai g2 spirit twin, by Thermo Fisher Scientific (2 mentions)
Hellmanex solution, by Merck Group (1 mentions)
Zetasizer nano series zs instrument, by Malvern Panalytical (1 mentions)
Nano s dynamic light scattering system, by Malvern Panalytical (1 mentions)
Zetasizer nano s system, by Malvern Panalytical (1 mentions)
Amicon ultra 4 centrifugal filter unit, by Merck Group (1 mentions)
7 protocols using 0.1 μm filter
1
SAXS Analysis of RNA Conformations
2
Monitoring ASC Pyrin Domain Fibrillization
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Immediately before the experiments, samples of monomeric soluble ASCPYD were centrifuged at 20,000 g at 4 °C for 30 min and filtered with 0.1 μM filter (Millipore). The protein concentration was adjusted to 25 μM by dilution from a higher-concentrated stock solution. Filament formation was triggered by rapid dilution to neutral pH. Thereby, 70 μl of monomeric ASCPYD was mixed with 0.45 μl of 2.75 M NaOH solution to a reach the pH of 7.5. The solution was mixed at room temperature by careful pipetting, to avoid introduction of air bubbles, and immediately transferred to a quartz cuvette with 1 cm path length. Between runs, cuvettes were carefully cleaned with 1 M Hellmanex solution (Sigma-Aldrich) to avoid cross-seeding effects between sequential measurements. Filament growth was monitored by dynamic light scattering with a Malvern Zetasizer Nano ZS series instrument. The laser focal spot was positioned in the middle of the cuvette and maintained fixed for all the measurements. To maximize the intensity of the scattered light, the minimal attenuation level was used. Data were acquired in 60 s intervals by averaging three runs of 20 s, until a total time of 350 min. Afterwards, the protein solution was blotted on EM grids, negatively stained and imaged with transmission electron microscopy to visualize filament formation.
3
Nanoparticle Size Characterization by DLS
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DLS data were collected on a Nano-S Dynamic Light Scattering system (Malvern Instruments Ltd., Malvern, UK) as previously reported [44 (link)]. Samples were filtered through a 0.1-μm filter (Millipore) and equilibrated for 5 minutes at 20°C before measurements. 15 measurements were made per sample, and for each condition three independent samples were tested.
4
Dynamic Light Scattering Analysis of RNA
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DLS data for RNA was collected prior to SAXS data collection to confirm that all the samples were highly pure and suitable for data collection. Samples were dialyzed for 2 hours at 4°C against Buffer 1 [50 mM Tris (pH 7.50), 100 mM NaCl, 5 mM MgCl2] prior to DLS analysis. After dialysis, samples were subjected to filtration through a 0.1μm filter (Millipore, USA) and equilibrated at 20°C. The hydrodynamic radius rH and homogeneity (distribution of rH) of each sample was examined using the Zetasizer Nano S system (Malvern Instruments Ltd., Malvern, UK) equipped with a 4 mW laser (λ = 633 nm) as previously described [24 (link), 25 (link), 31 (link)]. DLS experiments for each sample were performed at multiple concentrations: 2.20–3.70 mg/mL for wt, 1.00–1.40 mg/mL for Δloop, 0.40–0.80 mg/mL for Δbulge, 1.50–3.10 mg/mL for ΔCD, 0.70–1.30 mg/mL for ΔGU, 1.80–2.80 mg/mL for Δ2bp, 0.40–0.80 mg/mL for Δ4bp, 1.10–1.70 mg/mL for wt-PKR1-169 complex, and 1.00–1.50 mg/mL for Δloop-PKR complex. DLS data were analyzed using the DTS software (Version 6.01, Malvern Instruments Ltd., Malvern, UK).
5
Visualizing DNA Nanoparticles by TEM
6
Gastric Epithelial Cell OMV Exposure
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For preparation of culture supernatant (CM) from primary human gastric epithelial cells stimulated with H. pylori OMV, gastric epithelial cells were exposed to OMVs (200 μg/mL) for 24 h and then the CM was collected. To minimize the effect of any potentially contaminating OMVs, the CM was filtered using two sequential filters, first a 0.1 μm filter (Merck Millipore, Billerica, MA, USA), and then an Amicon Ultra-4 centrifugal filter unit with a molecular weight of 100 kDa (Merck Millipore). The resultant filtered CM was designated as “OMV-CM.” The medium obtained from primary human gastric epithelial cells cultured for 24 h in the absence of OMVs was designated as “control-CM.” In some experiments, primary human gastric epithelial cells were exposed to OMV-CM (50% v/v) or control-CM (50% v/v) for 24 h and then washed in PBS two times. After these washes, human eosinophils were cocultured with either OMV-CM-exposed or control-CM-exposed gastric epithelial cells for 24 h.
7
Visualizing DNA Nanoparticles by TEM
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DNA-NPs were visualized by transmission electron microscopy (TEM) using grids prepared as described previously with minor modifications48 (link). Briefly, carbon supported grids with copper mesh (CF200H-CU; Electron Microscopy Sciences) were glow discharged and soaked in 100 μM MgCl2 and blotted prior to depositing DNA-NPs. 20 μl of a 10 nM DNA-NP solution was applied to a clean parafilm surface and the grid was floated for 2 minutes. While soaking, 2% uranyl formate (UF; Electron Microscopy Sciences) was neutralized with 25 mM NaOH final concentration, vortexed for 1 minute, and filtered via syringe through a 0.1 μm filter (EMD Millipore) dropwise onto the clean parafilm surface. The grid was then removed and quickly dried by edge blotting with Whatman 44 ashless paper. The grid was then immediately transferred to the 2% UF solution and incubated for 30 seconds. Again, the grid was dried by blotting along the edge with Whatman paper, and left to dry in air for an additional 30 minutes prior to imaging. Imaging was done on a FEI Tecnai G2 Spirit TWIN set to 120kV equipped with a Gatan camera. Images were acquired at 6,500x for wide-field views and 52,000x for near-field views. Images were collected using 3-second exposures. All raw images were cropped in Adobe Photoshop with subsequent autocontrast applied.
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