C-flat™ holey carbon grids CFT-2/2–2C washed with chloroform overnight were glow discharged for 15 seconds at 5 mA immediately before the application of the sample. For each grid, a volume of 3.6 μL of the sample was deposited in the grid. All samples were applied to the grids without further dilution and at the concentration indicated in the preparation method described above. Vitrification was performed in a Vitrobot Mark IV (ThermoFisher) by blotting the grids for 3 seconds at a blot force of +2. The blotting chamber in the Vitrobot was set at a relative humidity of 100% and a temperature of 25 °C. Subsequently, grids were plunged into liquid ethane cooled down to near liquid nitrogen temperature. The cryo-TEM images were acquired on a Tecnai F20 electron microscope operated at 200kV using a Gatan 626 single tilt cryo-holder. Images were collected in a Gatan Ultrascan 4000 4k x 4k CCD Camera System Model 895 at a nominal magnification 60,000. Images produced by this camera had a calibrated pixel size of 1.8Å / pixel). The total electron dose per image was ~50 e- /Å2. Images were collected using a defocus range from −2.7 to −3.5 μm. Images were prepared for figures using Adobe Photoshop program.
Ultrascan 4000 4k x 4k ccd camera system model 895
Manufactured by Ametek
The Ultrascan 4000 4k x 4k CCD Camera System Model 895 is a high-resolution imaging device designed for laboratory applications. It features a 4096 x 4096 pixel CCD sensor capable of capturing detailed images. The system is equipped with the necessary components to interface with a computer for image acquisition and processing.
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Lab products found in correlation
3 protocols using ultrascan 4000 4k x 4k ccd camera system model 895
1
Vitrification and Cryo-TEM Imaging
2
Characterization of Nanoparticle Formulations
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The average particle size of the nanoparticles was measured by Dynamic Light Scattering (DLS) using a Particle Size Analyzer (Brookhavens Instruments Corporation, NY, USA). The samples were diluted 1:20 with deionized water and measured at a scattering angle of 90⁰ and temperature of 25 ⁰C. The Polydispersity Index (PDI) estimated the size distribution of the nanoparticles. The zeta potential was measured by a Zeta Potential Analyzer (Brookhavens Instruments Corporation, NY, USA) using electrophoretic laser Doppler anemometry. The size and shape of the nanoparticles were examined by Transmission Electron Microscopy (TEM) (FEI Tecnai G2 Spirit Twin 120 kV Cryo-TEM, Gatan Ultrascan 4000 4k x 4k CCD Camera System Model 895).
The yield of the nanoparticles was measured by the UV-spectrophotometric method. A standard curve of HSA solution dissolved in Bradford reagent was used as a reference and absorbance was measured at 595 nm. To determine encapsulation efficiency, nanoparticles were spin concentrated using centrifugal filters with molecular weight cut off (MWCO) of 10,000 Da for eluting the non-encapsulated MRN into the collection tube. A standard curve of MRN in a mixture containing DDQ/Ethanol was used as a reference and absorbance was measured at 356 nm [32 ].
The yield of the nanoparticles was measured by the UV-spectrophotometric method. A standard curve of HSA solution dissolved in Bradford reagent was used as a reference and absorbance was measured at 595 nm. To determine encapsulation efficiency, nanoparticles were spin concentrated using centrifugal filters with molecular weight cut off (MWCO) of 10,000 Da for eluting the non-encapsulated MRN into the collection tube. A standard curve of MRN in a mixture containing DDQ/Ethanol was used as a reference and absorbance was measured at 356 nm [32 ].
3
Negative Staining of Assembled Protein Filaments
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McGill University. 10 µl of protein samples were deposited and adsorbed for 1 min onto carbon-coated grids that were glow discharged before the application of proteins. Grids were Glow discharged for 1-2 min in a vacuum evaporator (Edwards Vacuum Carbon Coater E306) before adding the protein samples to improve quality of sample analysis. Assembled NFL as well as SacsJ were diluted to 0.24 µg/µl and processed for imaging by TEM. Excess of protein was removed by wicking the edge of the grid on a piece of Whatman paper. The samples were then negatively stained for 1 min using 10 µl of 2% (w/v) uranyl acetate and examined with a Cryo-TEM (FEI Tecnai G2 Spirit Twin) (Thermo Fisher Scientific) using an accelerated voltage of 120 KV. Images were acquired with a CCD camera (Gatan Ultrascan 4000 4k x 4k CCD Camera System Model 895). The diameters of the assembled filaments were measured on enlarged TEM micrographs using ImageJ software (National Institute of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/).
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