Rea1 or the Rea1_ΔAAA2L-H2α deletion mutant in buffer B without glycerol were diluted to a final concentration of 45 nM. AMPPNP, ATP, or ADP were added to reach a final concentration of 3 mM. Negative-stain electron microscopy was performed on plasma-cleaned carbon film on 400-square-mesh copper grids (Electron Microscopy Sciences). 3 μl sample was applied to the grids that were subsequently stained with 2% (w/v) uranyl acetate. Data collection was done on an FEI tecnai G2 operated at 200kV and equipped with a Gatan Ultrascan 2K*2K CCD camera. Data were collected at ∼1 μm underfocus, with a pixel size of 3.629 Å and an estimated dose of 25 electrons / Å2 during 1 s exposures. SerialEM was used for semiautomatic data acquisition. Around 8000 particles per data set were manually picked and processed with Relion using standard procedures. In order to not bias the orientation of the linker with respect to the AAA+ ring, the initial model consisted of the low pass filtered NTD-AAA+ ring map rescaled to the correct pixel size.
400 square mesh copper grids
Manufactured by Electron Microscopy Sciences
Sourced in United States
400-square-mesh copper grids are a type of lab equipment used in electron microscopy. They provide a stable and uniform support structure for samples being observed under an electron microscope. The copper material and square mesh pattern offer durability and consistent spacing to facilitate high-quality imaging and analysis.
Automatically generated - may contain errors
3 protocols using 400 square mesh copper grids
1
Structural Analysis of Rea1 AAA+ ATPase
2
Negative Stain EM of Recombinant Human Dynactin
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For negative stain EM, 400-square-mesh copper grids (Electron Microscopy Sciences) were plasma cleaned, and 3 μl of protein (≈100 nM in GF150 buffer) were applied to the grid. 20 μl of 2% (wt/vol) uranyl acetate were added to the sample, and excessive stain was removed with a filter paper. Micrographs were recorded on an FEI Tecnai G2 Spirit (120 kV) transmission electron microscope equipped with a Gatan Ultrascan 1000 XP CCD detector. Image acquisition was performed at 260,00× nominal magnification and 1.5 µm underfocus. For 2D classification of recombinant human dynactin, ∼5,000 particles were picked semi-automatically using EMAN2 (Tang et al., 2007 (link)) and 2D-classified using RELION 2.1 (Scheres, 2012 (link)).
3
TEM Imaging of Diluted Nanoparticles
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Sample preparation for TEM Diluted NP suspension in MilliQ ® water at a concentration of about 1 to 2 mg/ml were deposited on a carbon film of 400 square mesh copper grids (Electron Microscopy Sciences, Hatfield, PA USA). The volume of the droplet was about 2 to 4 µl. The droplet was allowed to sit for 5 minutes before excess of liquid was drained-out with filter paper.
Grids were allowed to dry in air for 1-2 hours before image acquisition. No staining procedure was introduced.
TEM imaging TEM image acquisition was done in bright field mode on a JEM-2100F, Field Emission electron microscope (Jeol Ltd, Tokyo, Japan) equipped with a sample holder cooled by liquid nitrogen (Gatan inc. Warrendale, Pittsburg, PA, USA). The grids were maintained at -170 o C throughout the acquisition with a temperature controller (Smart Set Model 900 Cold Stage controller; Gatan inc. Warrendale, Pittsburg, PA, USA). The grids were introduced in the microscope column under vacuum. Liquid nitrogen was added to the sample holder and temperature recorded. The sample was exposed to the electron beam only after the temperature had reached -170 o C. The acceleration voltage was set at 200 kV. Images were digitally recorded at a low electron dose to prevent damage to the heat-sensitive particles (current densities were between 5 and 15 pA/cm 2 ).
Grids were allowed to dry in air for 1-2 hours before image acquisition. No staining procedure was introduced.
TEM imaging TEM image acquisition was done in bright field mode on a JEM-2100F, Field Emission electron microscope (Jeol Ltd, Tokyo, Japan) equipped with a sample holder cooled by liquid nitrogen (Gatan inc. Warrendale, Pittsburg, PA, USA). The grids were maintained at -170 o C throughout the acquisition with a temperature controller (Smart Set Model 900 Cold Stage controller; Gatan inc. Warrendale, Pittsburg, PA, USA). The grids were introduced in the microscope column under vacuum. Liquid nitrogen was added to the sample holder and temperature recorded. The sample was exposed to the electron beam only after the temperature had reached -170 o C. The acceleration voltage was set at 200 kV. Images were digitally recorded at a low electron dose to prevent damage to the heat-sensitive particles (current densities were between 5 and 15 pA/cm 2 ).
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