Vitrobot mk3

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

The Vitrobot Mk3 is a laboratory instrument used for rapid vitrification of samples, a process that involves the rapid freezing of samples in a cryogenic environment to preserve their structure. The Vitrobot Mk3 automates the sample preparation process, allowing for consistent and reproducible results. It is designed to work with a variety of sample types and is commonly used in the fields of structural biology and cryo-electron microscopy.

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Vitrobot (424 citations)

5 protocols using vitrobot mk3

Cryo-TEM Imaging of CCMV-Avidin Complexes

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CCMV–avidin sample (15 mM NaCl) was prepared in the same way as for SAXS measurements and diluted 1: 9 with 15 mM NaCl. The sample was vitrified on a TEM grid and imaged at liquid nitrogen temperature. Vitrification was done using FEI Vitrobot Mk3 in a saturated water vapour environment. Sample volumes of 3 μl were placed on Quantifoil R 3.5/1 grids and the excess sample was blotted away with filter paper. Blot time and drain time were both 1 s. After blotting, the grids were plunged into liquid ethane/propane (1:1) solution that was cooled with liquid nitrogen surrounding the ethane/propane vessel. Vitrified samples were cryo-transferred to the microscope. Imaging was performed with JEOL JEM-3200FSC equipment operating at a 300-kV accelerating voltage and specimen temperature of ~86 K. Conventional TEM imaging of CCMV–avidin crystals functionalized with 5 nm AuNPs was carried out with Tecnai 12 Bio-Twin transmission electron microscope (FEI) using an acceleration voltage of 120 kV. The sample was added on Quantifoil R 3.5/1 grid and the excess sample was blotted away with filter paper. The functionalized CCMV–avidin–AuNP samples were imaged both with and without uranyl acetate (negative) staining.

Liljeström V., Mikkilä J, & Kostiainen M.A. (2014). Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins. Nature Communications, 5, 4445.

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Cryo-EM of 48S Ribosomal Complex

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Three μl of the 48S complex was applied to glow-discharged Quantifoil R2/2 cryo-EM grids covered with continuous carbon (of ~50 Å thick) at 4°C and 100% ambient humidity. After 30 s incubation, the grids were blotted for 2.5–3 s and vitrified in liquid ethane using a Vitrobot Mk3 (FEI).
Automated data acquisition was done using the EPU software (FEI) on a Tecnai F30 Polara G2 microscope operated at 300 kV under low-dose conditions (35 e^-/Å²) using a defocus range of 1.2–3.2 μm. Images of 1.1 s/exposure and 34 movie frames were recorded on a Falcon III direct electron detector (FEI) at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). Micrographs that showed noticeable signs of astigmatism or drift were discarded.

Llácer J.L., Hussain T., Saini A.K., Nanda J.S., Kaur S., Gordiyenko Y., Kumar R., Hinnebusch A.G., Lorsch J.R, & Ramakrishnan V. (2018). Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition. eLife, 7, e39273.

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Cryo-EM Imaging of 48S Complex

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3 μl of the 48S complex was applied to glow-discharged Quantifoil R2/2 cryo-EM grids covered with continuous carbon (of ∼50 Å thick) at 4°C and 100% ambient humidity. After 30 s incubation, the grids were blotted for 2.5–3 s and vitrified in liquid ethane using a Vitrobot Mk3 (FEI). Automated data acquisition was done using the EPU software (FEI) on a Titan Krios microscope operated at 300 kV under low-dose conditions (30 e^–/Å²) using a defocus range of 1.2–3.2 μm. Images of 1.1 s/exposure and 34 movie frames were recorded on a Falcon III direct electron detector (FEI) at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). More than 3000 micrographs (Supplementary Figure S1A) were recorded from two independent data acquisition sessions. Micrographs that showed noticeable signs of astigmatism or drift were discarded.

Llácer J.L., Hussain T., Dong J., Villamayor L., Gordiyenko Y, & Hinnebusch A.G. (2021). Large-scale movement of eIF3 domains during translation initiation modulate start codon selection. Nucleic Acids Research, 49(20), 11491-11511.

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Cryo-EM Grid Preparation and Imaging

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3 μl of each complex were applied onto glow-discharged Quantifoil R2/2 cryo-EM grids covered with continuous carbon (of around 50 Å thick) at 4°C and 100% ambient humidity. After a 30 s incubation, the grids were blotted for 3-3.5 s and vitrified in liquid ethane using a Vitrobot Mk3 (FEI).
Automated data acquisitions (EPU software, FEI) were done on Tecnai F30 Polara and Titan Krios microscopes (FEI) at 300 kV for the Sample1 dataset and the Sample2 (IF2-containing dataset), respectively. For the Sample1 dataset, images of 1.1 s/exposure and 17 movie frames were recorded on a Falcon III direct electron detector (FEI) at a calibrated magnification of 104,478 (yielding a pixel size of 1.34 Å). For the Sample2 dataset, images of 1.5 s/exposure and 25 movie frames were recorded on a Falcon II direct electron detector (FEI) at a calibrated magnification of 104,478, resulting in a pixel size of 1.34 Å. For both datasets, dose rates of 27-30 electrons per Å² per second and ranges from 1.5 to 3.0 μm defocus values were used. Micrographs that showed noticeable signs of astigmatism or drift were discarded.

Hussain T., Llácer J.L., Wimberly B.T., Kieft J.S, & Ramakrishnan V. (2016). Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation. Cell, 167(1), 133-144.e13.

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3D Reconstructions of Suilysin Prepore and Pore

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For 3D reconstructions of the prepore and the pore, 1 μl of 0.3–0.5
mg/ml solution of either wild-type or disulphide-locked suilysin was incubated
with 1 μl of liposomes for 10 min at 37°C. Liposomes were then applied
to lacey carbon-coated copper grids (Agar Scientific, Stansted, United Kingdom)
and frozen using Vitrobot Mk3 (FEI) at 22°C and 100% humidity. Images were
collected on a Tecnai G2 Polara microscope (FEI) at 300 kV, on a Gatan 4k ×
4k CCD camera giving a final pixel size of 2 Å, at an electron dose of
20–25 e/Å².

Leung C., Dudkina N.V., Lukoyanova N., Hodel A.W., Farabella I., Pandurangan A.P., Jahan N., Pires Damaso M., Osmanović D., Reboul C.F., Dunstone M.A., Andrew P.W., Lonnen R., Topf M., Saibil H.R, & Hoogenboom B.W. (2014). Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin. eLife, 3, e04247.

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