Aggregated proteins from the microfluidic device were obtained by excising the PDMS block from the glass slides using a sterile scalpel and flushing the surfaces repeatedly with 100 μl of ultrafiltered deionized water. The collected liquid was refrigerated along with the remaining liquid in the reservoirs until further analysis. Procedures for collecting aggregated proteins from shaking experiments as well as for EM sample preparation were described previously7 (link). Negative stain EM data were collected on a JEOL JEM1400 electron microscope with a Gatan Orius 832 CCD. Cryo-EM data were collected on a JEOL JEM-2100F microscope with K2 Summit direct detector. Data were processed using EMAN259 (link) and RELION-445 (link) software packages.
Orius 832 ccd
Manufactured by Ametek
Sourced in United States
The Orius 832 CCD is a charge-coupled device (CCD) camera designed for laboratory applications. It captures high-resolution images and operates with a resolution of 3296 x 2472 pixels. The Orius 832 CCD provides a sensor size of 23.4 mm x 17.6 mm and a pixel size of 7.4 μm x 7.4 μm. The camera is capable of delivering a frame rate of up to 16 frames per second.
Automatically generated - may contain errors
5 protocols using orius 832 ccd
1
Microfluidic Protein Aggregation Analysis
2
Freeze Fracture Analysis of Claudin Mutants
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HEK293T and COS7 cells were prepared for freeze fracture as previously described58 (link). Cells were fixed in 2% glutaraldehyde in PBS for 1 h and slowly transitioned to 30% glycerol as cryoprotectant. Cells were lifted with a cell scraper followed by “slam-freezing” using a LifeCell CF-100 equipped with sapphire disk cooled to − 186 °C as the freezing surface. Samples were fractured using a Balzer freeze-fracture apparatus and the replicas created by deposition of platinum at 45° followed carbon at 90° angles relative to the sample. The replicas were imaged using a JEOL 2100 TEM operating at 200 kV with a Gatan Orius 832 CCD. The SerialEM/Etomo software suite59 (link) was used to both collect and analyze the data. At least two independent replicas were analyzed for every Cldn mutant. P-face persistence length was determined by measuring the length of 50 P-face segments per Cldn. Strand breaks were quantified by tracing 0.8–1.5 µm lengths of 10 randomly selected strands from at least 2 replicas for each condition (n = 10) and counting the number of disruptions in strand P-face segregation along the length of the strand.
3
Negative Staining of Bacterial Cells for TEM
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For electron microscope, KTU-TGVF or KT2440 cells were negatively stained with 0.05% (w/v) uranyl acetate for 20 s on 200-mesh carbon-coated copper grids. Electron microscope images were captured using a JEOL JEM-1400 transmission electron microscope (TEM) (JEOL, Japan) operated at 80 kV and equipped with a GATAN Orius 832 CCD (GATAN, USA).
4
Protein Extraction and Cryo-EM Analysis
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Aggregated proteins from the microfluidic device were obtained by excising the PDMS block from the glass slides using a sterile scalpel and flushing the surfaces repeatedly with 100 μl of ultrafiltered deionized water. The collected liquid was refrigerated along with the remaining liquid in the reservoirs until further analysis. Procedures for collecting aggregated proteins from shaking experiments as well as for EM sample preparation were described previously (7 ). Negative stain EM data were collected on a JEOL JEM1400 electron microscope with a Gatan Orius 832 CCD. Cryo-EM data were collected on a JEOL JEM-2100F microscope with K2 Summit direct detector. Data were processed using EMAN2 (61 (link)) and RELION-4 (46 ) software packages.
5
Negative Staining of EBOV NP
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The EBOV NPΔ451–739 protein preparation was pre-treated with all kinds of conditions as required and diluted to 0.1 mg/mL in 0 or 150 mmol/L NaCl buffer. Then 5 μL sample was applied to 400 mesh copper grids coated with continuous carbon film, which had been plasma cleaned by glow charge, and negative-stained with 1% uranyl acetate. After air drying, the sample was observed on a JEOL-1400 EX electron microscope equipped with a Gatan Orius 832 CCD camera, operated at an acceleration voltage of 120 kV. For helical NLC particle data acquisition, the image was recorded on a magnification at a calibrated pixel size of 1.97 Å and defocus range of 2–6 μm.
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