The human proteasome α7 subunit (PSMA3 [P25788]; residues 1–255) was expressed and purified as described previously [23 (link),24 (link)]. The sample was dissolved in a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. An aliquot (2.5 μL) of sample solution was applied onto R1.2/1.3 MO 200 mesh holey grids (Quantifoil Micro Tools, Jena, Germany) coated with thin carbon membranes and pre-treated with glow discharge using a plasma ion bombarder (PIB-10, Vacuum Device, Mito, Japan) for 30 s. The grid was blotted for 4 s with a force level of 7 at 4 °C and 95% humidity, and then it was flash frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fischer Scientific, Hillsboro, OR, USA). The vitreous ice sample grid was maintained at liquid-nitrogen temperature within a JEM2200FS electron microscope (JEOL Ltd., Tokyo, Japan) using a side-entry Gatan 626 cryo-transfer holder (Gatan Inc., Pleasanton, CA, USA), and it was imaged using a field-emission gun operated at 200 kV and an in-column (Omega-type) energy filter operating in zero-energy-loss mode with a slit width of 20 eV. A total of 100 images were collected on a direct-detector CMOS camera (DE20, Direct Electron, LP, San Diego, CA, USA) at a nominal magnification of 40,000×, corresponding to 1.42 Å per pixel on the specimen.
Gatan 626 cryo transfer holder
Manufactured by Ametek
Sourced in Japan
The Gatan 626 cryo-transfer holder is a specialized piece of lab equipment designed for the transfer of cryogenically-cooled samples in a transmission electron microscope (TEM) environment. It enables the transportation and observation of samples maintained at low temperatures to preserve their structural integrity.
Automatically generated - may contain errors
Lab products found in correlation
Jem 2200fs electron microscope, by JEOL (1 mentions)
Vitrobot mark 4 system, by Thermo Fisher Scientific (1 mentions)
Nahco3, by Merck Group (1 mentions)
Na2co3, by Merck Group (1 mentions)
Multi a holey carbon film, by Quantifoil (1 mentions)
Cryo box, by Zeiss (1 mentions)
Emmanu4 v, by TVIPS (1 mentions)
3 protocols using gatan 626 cryo transfer holder
1
Structural Analysis of the Human Proteasome
2
Biomineralization and Coacervation Synthesis
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CaCl2, Na2CO3 (Sigma), and NaHCO3 (Sigma) powders were dissolved in 10 mM tris (pH 8) and used for CaCO3 mineralization at a final concentration of 10, 8, and 2 mM, respectively. The measured pH of the resulting mineralization solution was approximately 8.8. For simultaneous mineralization and coacervation, rPif80 was homogeneously mixed with Na2CO3/NaHCO3 solutions, and then, the CaCl2 solution was added to the mixture. The final concentration of rPif80 was 1 g/liter. Control experiments were performed without rPif80 or with lysozyme-HA complex coacervate (50 (link)). For complex coacervation, lysozyme and HA were used at concentrations of 3 and 1 g/liter, respectively. After the reaction, the samples were incubated at 4°C for further analysis.
The resulting CLP was observed by phase-contrast optical microscopy, and grown mineral precipitates on an Si wafer were observed by optical microscopy (CK30, Olympus) and analyzed by confocal Raman microscopy (alpha300 RA Plus, WITec) to determine the polymorph of the mineral. To investigate the inside of the liquid phase, aliquots of the suspensions were blotted on a grid and quickly frozen for microscopic analysis, electron diffraction patterns, and EDS performed with cryo-TEM (Libra 200 HT MC Cs, Carl Zeiss), using a Gatan 626 cryo transfer holder (Gatan) and operating at approximately −180°C.
The resulting CLP was observed by phase-contrast optical microscopy, and grown mineral precipitates on an Si wafer were observed by optical microscopy (CK30, Olympus) and analyzed by confocal Raman microscopy (alpha300 RA Plus, WITec) to determine the polymorph of the mineral. To investigate the inside of the liquid phase, aliquots of the suspensions were blotted on a grid and quickly frozen for microscopic analysis, electron diffraction patterns, and EDS performed with cryo-TEM (Libra 200 HT MC Cs, Carl Zeiss), using a Gatan 626 cryo transfer holder (Gatan) and operating at approximately −180°C.
3
Cryogenic EM Imaging of Extracellular Vesicles
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EVs isolated from 4 × 104 infected monocytes (MOI 1:1) were collected in 4-µL suspension, and were applied onto copper EM-grids covered by a QUANTIFOIL Multi A holey carbon film (Quantifoil Micro Tools), and excess of liquid was blotted automatically between two strips of filter paper. Subsequently, the samples were rapidly plunge-frozen in liquid ethane (cooled by liquid nitrogen at about −180 °C) in a cryobox (Carl Zeiss). Excess of ethane was removed with a piece of filter paper. The samples were transferred immediately using a Gatan 626 cryo-transfer holder (Gatan) into a pre-cooled cryo-electron microscope (Philips) operated at 120 kV and imaged under low dose conditions. The images were recorded with a 2k F216-CMOS-camera (CMOS-camera and acquisition software, EMMANU4 v 4.00.9.17, TVIPS). In order to minimize the noise, four images were recorded and averaged to one image.
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