For negative staining, 2.5 µl of the purified proteasome without or with its regulator (~0.01–0.1 mg/ml) was applied to glow-discharged EM grids covered by a thin layer of continuous carbon film (TED Pella, Inc.) and stained with 0.75% (w/v) uranyl formate solution as described36 (link). EM grids were imaged on a Tecnai T20 microscope (Thermo Fisher Scientific) operated at 200 kV with a TVIPS TemCaM F816 scintillator-based CMOS camera (TVIPS). Images were recorded at a magnification of ×50,000, which resulted in a 3.139-Å pixel size on the specimen. Defocus was set to −1.5 µm to −2 µm. For cryo-EM, 2.5 µl of purified proteasome sample (~1.0 mg/ml) was applied to glow-discharged holy carbon grids (Quantifoil 400 mesh Cu R1.2/1.3). For PAN-T20S sample, 2.5 μl (~0.1 mg ml−1) was applied onto amino-functionalized graphene oxide holy carbon grids (Quantifoil 300 mesh Au R1.2/1.3). The grids were blotted by Whatman No. 1 filter paper and plunge-frozen in liquid ethane using a Mark III Vitrobot (Thermo Fisher Scientific) with blotting times of 8–12 s at room temperature and over 90% humidity. Cryo-EM datasets were collected using SerialEM in three different microscopes all equipped with Field Emission source and K2 or K3 camera (Gatan Inc.) operated in super-resolution mode (Supplementary Table 3 ).
Continuous carbon film
Manufactured by Ted Pella
Sourced in United States
Continuous carbon film is a thin, uniform carbon coating used as a support film in transmission electron microscopy (TEM) sample preparation. It provides a stable and clean surface for mounting and supporting delicate samples during TEM analysis.
Automatically generated - may contain errors
Lab products found in correlation
Temcam f816, by TVIPS (1 mentions)
Tecnai t20 microscope, by Thermo Fisher Scientific (1 mentions)
1400 flash, by JEOL (1 mentions)
Oneview camera, by JEOL (1 mentions)
Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions)
Tecnai t12 microscope, by Thermo Fisher Scientific (1 mentions)
Oneview 4k camera, by Ametek (1 mentions)
Jem 1400plus, by JEOL (1 mentions)
Ribolock, by Thermo Fisher Scientific (1 mentions)
5 protocols using continuous carbon film
1
Negative Staining and Cryo-EM of Proteasomes
2
Transmission Electron Microscopy of Nanostructures
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A 3 μL droplet
of nanostructures/uranyl acetate mixture solution was applied to the
copper grid with continuous carbon film (Ted Pella, Inc.) The droplet
was blotted using a filter paper from the edge of the grid. Images
were obtained from a grid with dried nanostructures using the JEOL-1400Flash
(JEOL, Inc. Japan) with a Gatan Oneview camera at room temperature.
of nanostructures/uranyl acetate mixture solution was applied to the
copper grid with continuous carbon film (Ted Pella, Inc.) The droplet
was blotted using a filter paper from the edge of the grid. Images
were obtained from a grid with dried nanostructures using the JEOL-1400Flash
(JEOL, Inc. Japan) with a Gatan Oneview camera at room temperature.
3
Negative Staining and Cryo-EM of Skd3 Protein
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For negative staining, 2.5 μl of the purified Skd3 (0.1 mg/ml) was applied to glow-discharged EM grids covered by a thin layer of continuous carbon film (TED Pella Inc.) and stained with 0.75% (w/v) uranyl formate solution. EM grids were imaged on a Tecnai T12 microscope (Thermo Fisher Scientific) operated at 120 kV with Gatan Rio Camera. Images were recorded at a magnification of ×52,000, resulting in a 2.2-Å pixel size on the specimen. Defocus was set to −1.5 to −2 μm.
For cryo-EM, 5 to 6 μl of purified PARLSkd3_WB (1.06 mg/ml) were applied to glow-discharged holy carbon grids (Quantifoil 300 mesh Au R1.2/1.3). Four microliters of PARLSkd3_WB (0.25 mg/ml) was applied onto holy carbon grids (Quantifoil 300 mesh Au R1.2/1.3) coated with PEG-amino–functionalized graphene oxide. The grids were blotted by Whatman no. 1 filter paper and plunge-frozen in liquid ethane using Mark IV Vitrobot (Thermo Fisher Scientific) with blotting times of 3 to 6 s at room temperature and over 90% humidity.
Cryo-EM datasets were collected at Stanford-SLAC Cryo-EM Center using EPU2.9 in Titan Krios G3i equipped with energy filter Selectris and Falcon4. From Quantifoil grids, 10,723 micrographs of no tilt data and 2151 micrographs of the tilt data were pooled to form dataset 1. From the PEG-amino grids (56 (link)), 10,854 micrographs were collected without tilt in dataset 2.
For cryo-EM, 5 to 6 μl of purified PARLSkd3_WB (1.06 mg/ml) were applied to glow-discharged holy carbon grids (Quantifoil 300 mesh Au R1.2/1.3). Four microliters of PARLSkd3_WB (0.25 mg/ml) was applied onto holy carbon grids (Quantifoil 300 mesh Au R1.2/1.3) coated with PEG-amino–functionalized graphene oxide. The grids were blotted by Whatman no. 1 filter paper and plunge-frozen in liquid ethane using Mark IV Vitrobot (Thermo Fisher Scientific) with blotting times of 3 to 6 s at room temperature and over 90% humidity.
Cryo-EM datasets were collected at Stanford-SLAC Cryo-EM Center using EPU2.9 in Titan Krios G3i equipped with energy filter Selectris and Falcon4. From Quantifoil grids, 10,723 micrographs of no tilt data and 2151 micrographs of the tilt data were pooled to form dataset 1. From the PEG-amino grids (56 (link)), 10,854 micrographs were collected without tilt in dataset 2.
4
TEM Imaging of Biomolecular Samples
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For TEM analysis, 3 μL of samples at 15 μM were applied to glow-discharged (15 mA, negative charge for 25 s), 400-mesh copper grids covered by a thin layer of continuous carbon film (Ted Pella). After 1 min, the excess sample was removed using a filter paper. Uranyl acetate solution (2%) was applied twice to the grids for 30 sec each and the excess solution was removed. Specimens were analyzed with a defocus range of −1 to −2 μm, using a Jeol JEM-1400Plus instrument operating at 120 kV. Images were acquired using an OneView 4K camera (Gatan). Sample preparation and data acquisition were performed at the Electron Microscopy Laboratory (LME) at Brazilian Nanotechnology National Laboratory (LNNano).
5
Structural Characterization of CsPABPN1-Poly(A) Interaction
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CsPABPN1 purified by affinity and gel-filtration chromatographies, at a 30 M final concentration, was incubated on ice for 2 h in binding buffer containing 10 mM Tris-HCl, pH 7.5, 7 M MgCl 2 , 0.1 M EDTA, 0.6 M DTT and 20 U Ribolock (Thermo Scientific, USA), in the presence and absence of 1.0 M poly(A 50 ) (Invitrogen, USA). CsPABPN1-poly(A 50 ), CsPABPN1 or poly(A 50 ) alone were applied to glowdischarged (15 mA, negative charge for 25 seconds) 400 mesh copper grids covered by a thin layer of continuous carbon film (Ted Pella Inc., USA). After 1 min, the excess sample was blotted using a filter paper and the grid washed three times using the binding buffer. Uranyl acetate solution (2%) was applied twice to the grids for 30 seconds and the excess solution was blotted. Images were collected at room temperature in a Jeol JEM-2100 microscope operated at 200 kV and recorded on a TemCam F-416 4k 4k CMOS camera (Tietz Video and Image Processing Systems, Germany), using a nominal magnification of 60,000 and 100,000 in a defocus range of 1 to 3 µm.
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