Mk 3 vitrobot

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Mk III Vitrobot is a laboratory instrument used for the automated preparation of samples for cryo-electron microscopy. It is designed to rapidly flash-freeze biological specimens in a controlled environment, preserving their native structure for high-resolution imaging.

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Lab products found in correlation

Ultrascan 4000 ccd camera, by Ametek (2 mentions) Krios electron microscope, by Ametek (1 mentions) K2 summit detector, by Ametek (1 mentions) R2 1 grid, by Quantifoil (1 mentions) Cm10 microscope, by Philips (1 mentions) Tecnai f20 microscope, by Ametek (1 mentions) 626 cryo holder, by Ametek (1 mentions)

Spelling variants of this product

Vitrobot (424 citations) Vitrobot III (6 citations)

4 protocols using mk 3 vitrobot

Actin Filament Decoration by MyoA:ELC:MTIP Complex

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MyoA:ELC:MTIP and Act1 were expressed and purified as previously described [10 (link),24 (link)]. For Act1 samples, ammonium acetate was removed by a spin column, and Act1 (13.1 μM) was polymerized in the presence of 13 μM jasplakinolide by adding 10X KMEI, in final 1x concentration of 10 mM Hepes, pH 7.5, 50 mM KCl, 4 mM MgCl2, 1 mM EGTA. Polymerized filamentous Act1 was diluted down to 0.13 μM, and treated with apyrase (77 μg/ml) for 20 min before addition of the MyoA:ELC:MTIP complex in a 1:1 ratio.
For Act1-only samples, no apyrase was used. After 30 min incubation, negatively stained or cryo-EM grids were prepared. The quality of the decorated filaments was first checked with negative staining, and after confirming the presence of decorated Act1 filaments, frozen-hydrated samples were prepared on Quantifoil 2/2, 200 mesh in-air glow-discharged grids using an Mk III Vitrobot (FEI). 3 μl of sample was applied on a grid and blotted for 3 s before plunge-freezing in liquid ethane.

Vahokoski J., Calder L.J., Lopez A.J., Molloy J.E., Kursula I, & Rosenthal P.B. (2022). High-resolution structures of malaria parasite actomyosin and actin filaments. PLoS Pathogens, 18(4), e1010408.

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Cryo-EM Imaging of Influenza Hemagglutinin

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Grids of full-length HA were plunge-frozen using an Mk III Vitrobot (FEI) at a protein concentration of ∼2 mg/mL, supplemented with 0.1% octyl-β-glucoside (OG) to reduce orientational bias. Then 4 μL of sample was applied to glow-discharged 300-mesh copper R2/4 quantifoil grids and blotted for 5 s before plunging into liquid ethane. Grids of HA−Fab complex were frozen using an Mk IV Vitrobot with protein at a concentration of ∼1.5 mg/mL also supplemented with 0.1% OG. Then 4 uL of sample was applied to glow-discharged 200-mesh copper R2/2 quantifoil grids and blotted for 3 s before plunge-freezing. Both specimens were imaged using a Titan Krios electron microscope operating at 300 kV. Micrographs were recorded in counting mode using a Gatan K2 Summit detector mounted at the end of a Gatan GIF Quantum energy filter operating in zero-loss mode with a slit width of 20 eV. Exposures were 8 s, with a total dose of 43.7 e/Å² fractionated into 20 frames with a calibrated pixel size of 1.08 Å. Images were recorded with a defocus of 1 µm to 4 µm.

Benton D.J., Nans A., Calder L.J., Turner J., Neu U., Lin Y.P., Ketelaars E., Kallewaard N.L., Corti D., Lanzavecchia A., Gamblin S.J., Rosenthal P.B, & Skehel J.J. (2018). Influenza hemagglutinin membrane anchor. Proceedings of the National Academy of Sciences of the United States of America, 115(40), 10112-10117.

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Cryo-EM Structural Analysis of Viral Capsids

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Three microliters of purified capsids were pipetted onto a freshly glow-discharged Quantifoil R2/1 grid (Quantifoil Micro Tools GmbH, Jena, Germany) and mounted in an FEI Vitrobot Mk III (FEI, Hillsboro, OR, USA) for blotting and plunge-freezing into a 60:40 mix of liquid ethane/propane. Grids were transferred onto a Gatan 626 cryoholder and mounted into an FEI TF20 cryo-electron microscope (Hillsboro, OR, USA) maintaining liquid nitrogen temperature throughout. The microscope was operated at 200 kV and the sample was imaged using standard low-dose conditions at a magnification of 50,000× on a Gatan UltraScan 4000 CCD camera (Pleasanton, CA, USA) with a post-column magnification of 1.4×. The final pixel size corresponded to 2.1 Å at the sample. Image reconstruction was performed with the AUTO3DEM package [36 (link)] and the resolution estimated for the A- and B-capsid maps were 21 Å and 19 Å, respectively, according to the Fourier shell correlation limit of 0.5.

Tandon R., Mocarski E.S, & Conway J.F. (2015). The A, B, Cs of Herpesvirus Capsids. Viruses, 7(3), 899-914.

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Cryo-negative Staining of Phage Samples

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The phage samples for non-cryo TEM analysis were adsorbed onto glow-discharged carbon-coated grids, negatively stained with a 1% uranyl acetate solution, and examined on a Phillips CM-10 microscope at the Manawatu Microscopy and Imaging Centre (MMIC, Institute of Fundamental Sciences, Massey University).
Cryo-negative staining was performed at the University of Pittsburgh, Pittsburgh, PA, USA, on an FEI (Hillsboro, OR) Tecnai F20 microscope equipped with a Gatan (Pleasanton, CA, USA) 626 cryoholder and operated at 200 kV and nominal magnification of 50,000×. Briefly, 3 μL of sample were pipetted onto a holey carbon/copper grid that had been briefly glow-discharged. The grid was then placed sample-down onto a 100 μL droplet of 16% ammonium molybdate (in the pH range 7.0–8.0) and floated for 60 s, following the cryo-negative staining procedure (Adrian et al., 1998 (link)). The grid was then removed, blotted and plunge-frozen into liquid ethane using an FEI Vitrobot Mk III. Images were collected using standard low-dose techniques on a Gatan Ultrascan 4000 CCD camera with post-column magnification of 1.33×, yielding a pixel size at the sample of 2.3 Å.

Sattar S., Bennett N.J., Wen W.X., Guthrie J.M., Blackwell L.F., Conway J.F, & Rakonjac J. (2015). Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage. Frontiers in Microbiology, 6, 316.

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