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.
Hellmanex solution
Manufactured by Merck Group
Hellmanex solution is a laboratory cleaning and decontamination agent. It is designed to remove organic and inorganic contaminants from laboratory equipment and surfaces.
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2 protocols using hellmanex solution
1
Monitoring ASC Pyrin Domain Fibrillization
2
Lipoprotein Interaction with Lipid Bilayers
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Experiments were performed on a Q-Sense E4 quartz crystal microbalance (Q-Sense, Göteborg, Sweden). All experiments were measured at 37 °C in duplicate. Tubing, cells, and o-rings were cleaned first in 2% Hellmanex solution (Sigma Aldrich), rinsed in ultra-pure water and ethanol (99.9 %, Sigma Aldrich) before drying under nitrogen. Silicon oxide sensors were cleaned in the same way before UV-Ozone treatment for 10 minutes (BioForce Procleaner, Bioforce Nanosciences, Salt Lake City, USA), resulting in highly hydrophilic surfaces fully wettable by water (contact angles of less than 10°). Resonance frequencies were obtained in ultra-pure water and bilayers formed as described above using a flow rate of 100 µL min -1 until stable signals, characteristic for complete bilayers (∆F -25 s -1 , ∆D 0 a.u.), were established. [25] The bilayers were then washed in Tris buffer at 100 µL min -1 for 20 minutes. Subsequently, 1 mL of either HDL (0.132 mg mL -1 , based on protein content) or LDL (0.1 mg mL -1 based on protein content) was pumped into the measurement cell at 100 µL min -1 , whereafter the pump was stopped and the lipoproteins allowed to incubate for 12 hours at 37 °C. The sample was then washed for 30 minutes at 100 µL min -1 with Tris buffer and no change was detected upon rinsing.
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