Aβ42 monomers were purified the as described above for aggregation kinetics. The sample was diluted with water and highly concentrated NaCl solution was added to give samples with 10 μM Aβ42 monomer in sodium phosphate buffer with ionic strengths of 29 mM, 57 mM and 329 mM. The samples were loaded in a 96-well PEG-coated polystyrene plate (Corning 3881, USA) and ThT was added to one well at each ionic strength as the fluorescence control. Fibrils were formed in the same way as described as described above for SPR and 5 μl sample at each ionic strength was loaded as a liquid film on a lacey carbon filmed cooper grid. A layer of sample less than 300 nm thick was produced on the grid by blotting the extra liquid away at the back of the grid using a filter paper, followed by flash freezing the grid in liquid ethane and stored in liquid nitrogen. The grid preparation was carried out in a controlled environment vitrification system to ensure the stable temperature and humidity in order to maintain the original states of the sample. Images were recorded using a 120 kV electron microscope (Philips CM120 BioTWIN Cryo) with a CCD camera.
Cm120 biotwin cryo
Manufactured by Philips
The CM120 BioTWIN Cryo is a lab equipment product manufactured by Philips. It is designed for cryogenic applications, providing a stable and controlled environment for various research and experimental purposes. The product's core function is to enable precise temperature control and monitoring within a cryogenic setting, without further interpretation or extrapolation on its intended use.
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Lab products found in correlation
4 protocols using cm120 biotwin cryo
1
Aβ42 Fibril Formation Kinetics at Varying Ionic Strengths
2
Cryo-TEM analysis of Amyloid-β aggregation
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Monomeric Aβ42 peptide was isolated in 20 mM sodium phosphate buffer, 200 μM EDTA, 0.02% sodium azide, pH 8.0 by size-exclusion chromatography as described above. The sample was diluted with buffer and denaturant and ThT was added. The final concentrations were 1 M and 3 M of GuHCl or urea, or 1 M urea + 1 M NaCl, 9-10 μM Aβ42 and 1 μM ThT, pH 8.0. The aggregation kinetics were recorded using a Fluostar Optima plate reader (BMG Labtech) at 37°C under quiescent conditions. Samples for cryo-TEM were taken once the aggregation reaction had reached the fluorescence plateau. Five microliters were loaded on a lacey carbon filmed copped grid and flash frozen in liquid ethane at −180°C. The frozen samples were stored in liquid nitrogen and images were taken by a 120-kV electron microscope (Philips CM 120 BioTWIN Cryo). Alternatively, a Fischione Model 2550 cryo transfer tomography holder was used to transfer the specimen into the electron microscope (JEM 2200FS) equipped with an in-column energy filter (Omega filter), which allows zeroloss imaging. The acceleration voltage was 200 kV for the JEM2200FS electron microscope and zero-loss images were recorded digitally with a TVIPS F416 camera using SerialEM under low dose conditions with a 30 eV energy selecting slit in place.
3
Cryo-TEM Analysis of Protein Fibrils
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Fibrils were collected after reaching the plateau phase in the aggregation process and analysed by cryogenic transmission electron microscopy (TEM). The samples were loaded as a liquid film on a lacey carbon filmed cooper grid (01881F Lacey F/C, 200 mech Cu; PELCO No.160). A layer of sample less than 300 nm thick was produced on the grid by blotting the extra liquid away at the back of the grid using a filter paper, followed by flash freezing the grid in liquid ethane and whereafter it was stored in liquid nitrogen. The grid preparation was carried out in a controlled environment vitrification system to ensure the stable temperature and humidity in order to maintain the original state of the sample. Images were recorded using a 120 kV electron microscope (Philips CM120 BioTWIN Cryo) with a CCD camera. The size of the fibrils, for statistical purposes, were analysed using ImageJ (Schneider et al., 2012 (link)).
4
Cryo-TEM analysis of Aβ42 and Aβ5-42 aggregation
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Monomeric Aβ42 and Aβ5-42 were prepared as described above. Five different conditions were studied using cryo-TEM: Aβ5-42, coaggregation of Aβ42 and Aβ5-42, cross-seeding of Aβ42 and Aβ5-42 (with either Aβ42 or Aβ5-42 seeds) and self-seeding of Aβ5-42 and Aβ42. The total Aβ concentration was 10 µM and aggregation experiments were performed with and without ThT, at 37 °C, under quiescent conditions. A sample without ThT was taken when the aggregation reaction reached the plateau value and 5 µL were loaded to a thin film on a lacey carbon filmed copper grid, <300 nm thick, and flash frozen in liquid ethane at -180 °C and stored under liquid nitrogen. Images were acquired using a 120 kV electron microscope (Philips CM120 BioTWIN Cryo).
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