For standard lacey carbon grid (Ted Pella #01895-F) datasets, trimeric SARS-CoV-2 spike at a concentration of 1 mg/mL in 20 mM HEPES pH 7.5, 150 mM NaCl was combined with 1 molar equivalent of a papain-cleaved Fab form of either 2B04 or 2H04 in 20 mM HEPES pH 7.5, 150 mM NaCl, and incubated for 10 minutes before flash-freezing on lacey carbon grids using a Vitrobot Mk IV (ThermoFisher Scientific). For lacey carbon grids with ultra-thin carbon film (Ted Pella #01824G), SARS-CoV-2 spike was diluted to 0.2 mg/mL prior to mixing with 1 molar equivalent of either 2B04 or 2H04 Fab and then vitrified on thin-film lacey carbon grids using a Vitrobot Mk IV (ThermoFisher Scientific). Both sets of grids were glow discharged prior to sample application in a GloQube (EMS) for at least 20 s under vacuum.
Vitrobot mk 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Vitrobot Mk IV is a high-performance instrument designed for the preparation of cryo-electron microscopy (cryo-EM) samples. It is an automated plunge-freezing device that rapidly freezes samples in vitreous ice, enabling the preservation of their native structure and function. The Vitrobot Mk IV is a critical tool for researchers studying the structure and dynamics of biological macromolecules, such as proteins and protein complexes, using cryo-EM techniques.
Automatically generated - may contain errors
Lab products found in correlation
Gloqube system, by Quorum Technologies (7 mentions)
Talos arctica, by Thermo Fisher Scientific (5 mentions)
Titan krios, by Thermo Fisher Scientific (4 mentions)
K3 camera, by Ametek (4 mentions)
Ultraufoil 1.2 1 3 300 mesh grids, by Quantifoil (4 mentions)
C flat holey thick carbon grids, by Electron Microscopy Sciences (4 mentions)
Titan krios microscope, by Thermo Fisher Scientific (3 mentions)
Epu software, by Thermo Fisher Scientific (3 mentions)
A 10 nm colloidal gold, by Merck Group (2 mentions)
Tecnai tem 120 kev, by Thermo Fisher Scientific (2 mentions)
2k ccd, by Ametek (2 mentions)
626 cryo holder, by Ametek (2 mentions)
Lacey carbon 300 mesh gold grids, by Ted Pella (2 mentions)
Falcon 2 direct electron detector, by Thermo Fisher Scientific (2 mentions)
R2 1 grid, by Quantifoil (2 mentions)
Cryo electron microscopy grid, by Protochips (2 mentions)
Whatman no 4 filter paper, by Cytiva (2 mentions)
R1 2 1, by Quantifoil (2 mentions)
Cm12 electron microscope, by TVIPS (2 mentions)
1kx1k ccd camera, by TVIPS (2 mentions)
74 protocols using vitrobot mk 4
1
SARS-CoV-2 Spike Protein Complexes with Fab Fragments
2
Cryo-EM Sample Preparation for Viral Particles
3
Cryogenic Imaging of EV-F4 Capsids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An aliquot of 3.5 μL of purified EV-F4 full capsids at a concentration of 0.3 mg/mL was applied to Quantifoil R2/1 grids coated with 0.2 mg/mL of graphene oxide and previously glow discharged with a negative polarity at 15 mA for 15 s using a PELCO easiGlow. Samples were blotted with Whatman filter paper for 2.5 s before plunged into liquid ethane cooled with liquid nitrogen in a FEI Vitrobot MkIV. Vitrified samples were imaged using the FEI Titan Krios at 300 kV with a Gatan K3 Bioquantum camera and a nominal magnification of 105 kx. Movie frames were collected with a total dose of 41 e/Å2 over 30 frames with a final pixel size of 0.857 Å.
4
LASV L-Cstrep Polymerase Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LASV L-Cstrep protein with a concentration of 3 µM in assay buffer (100 mM HEPES(NaOH) pH 7.0, 50 mM NaCl, 50 mM KCl, 2 mM MnCl2 and 2 mM dithiothreitol) was mixed sequentially with single-stranded 5′ (0–19) vRNA and single-stranded 3′ (1–19) vRNA in 1.7-fold and primer C8 in 3.3-fold molar excess (all RNAs are listed in Supplementary Table 1 ). After 45 min incubation on ice, the reaction was started by addition of NTPs (0.25 mM GTP/ATP/UMPNPP and 0.125 mM CTP). After incubation at 30 °C for 2 h, 3 µL of the reaction was applied to glow-discharged Quantifoil R2/1 Au G200F4 grids, immediately blotted for 2 s using an FEI Vitrobot Mk IV (4 °C, 100% humidity, blotting force–10) and plunge frozen in liquid ethane/propane cooled to liquid nitrogen temperature.
5
Cryo-EM Imaging of Bacterial Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
PSY-grown aerobic cultures at an OD600 of 1 were concentrated 5 times and frozen in a Vitrobot MkIV (FEI, Hillsboro, Oreg.) as described previously [47, 48]. In brief, 2 μl of a 10 nm colloidal gold (Sigma Aldrich, St. Louis, Mo.) in 5% Bovine serum albumin (BSA) was added to 8 μl of culture. 3 μl of this suspension was placed onto a glow discharged carbon-coated R 2/2 Quantifoil copper-finder grid in the Vitrobot maintained at 22.5° C. with 95% humidity. This was followed by a 3 s blot with a pressure of 6 atm, a drain time of 1 sec, and plunge freezing in a mixture of liquid ethane (63%) and propane (37%). The frozen grids were then stored in liquid nitrogen until further use. Grids were imaged in Tecnai TEM 120 KeV (FEI, Hillsboro, Oreg.) at −178° C. using a Gatan 626 cryoholder and Gatan 2×2K CCD. Images were acquired with Digital Micrograph at 15,000× magnification [49, 50].
6
Cryo-EM Imaging of Bacterial Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 9
PSY-grown aerobic cultures at an OD600 of 1 were concentrated 5 times and frozen in a Vitrobot MkIV (FEI, Hillsboro, Oreg.) as described previously [47, 48]. In brief, 2 μl of a 10 nm colloidal gold (Sigma Aldrich, St. Louis, Mo.) in 5% Bovine serum albumin (BSA) was added to 8 μl of culture. 3 μl of this suspension was placed onto a glow discharged carbon-coated R 2/2 Quantifoil copper-finder grid in the Vitrobot maintained at 22.5° C. with 95% humidity. This was followed by a 3 s blot with a pressure of 6 atm, a drain time of 1 sec, and plunge freezing in a mixture of liquid ethane (63%) and propane (37%). The frozen grids were then stored in liquid nitrogen until further use. Grids were imaged in Tecnai TEM 120 KeV (FEI, Hillsboro, Oreg.) at −178° C. using a Gatan 626 cryoholder and Gatan 2×2K CCD. Images were acquired with Digital Micrograph at 15,000× magnification [49, 50].
7
Cryo-EM Analysis of Mec1-Ddc2 Complex
8
Cryo-TEM of Purified MrNV Capsid Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Purified MrNV capsid protein at approximately 1 mg/ml was prepared for cryogenic transmission electron microscopy using an FEI Vitrobot Mk IV. Four μl of VLP preparation was applied to freshly glow-discharged quantifoil holey carbon support films (R2/2; Quantifoil, Jena, Germany), blotted for 4 seconds and plunged into liquid ethane36 (link). Vitrified samples were viewed at low-temperature (around 98 K) and under low electron dose conditions using a JEOL 2200 FS cryo-microscope operated at 200 kV. Samples were held in a Gatan 626 cryo-stage. Micrographs were recorded at 50,000 × magnification on a Direct-Electron DE20 DDD camera giving a sample rate of 1.09 Å/pixel. Three-second exposures, at an electron dose rate of ~20 e/Å2/s, were captured in movie mode running at 20 frames per second.
9
Cryo-EM Imaging of Bacterial Membranes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Strains were grown aerobically in LB at 37°C until an OD600 of 0.6 was reached. Cells were spun for 5 min at 6,000 × g at 4°C and resuspended to an OD600 of about 12.
UltraAuFoil R2/2 grids (200 mesh; Quantifoil Micro Tools GmbH) were glow-discharged for 60 s at 10 mA. Cells were mixed with a solution of 10 nm colloidal gold (Sigma) immediately before freezing. A 2.5-μl droplet of sample was applied to the grid and plunge frozen using a Vitrobot MkIV (FEI Company) with a wait time of 60 s, a blot time of 5 s, a blot force of 3, and a drain time of 1 s at a constant humidity of 100%. Grids were stored under liquid nitrogen until required for data collection.
Projection images were collected on a 200 keV FEI Tecnai TF20 FEG transmission electron microscope (FEI Company) equipped with a Falcon II direct electron detector (FEI Company) using a Gatan 626 cryogenic-holder (Gatan). Leginon automated data-collection software 3.0 [41 (link)] was used to acquire images with pixel size of 0.828 nm (nominal magnification 25,000×) with a defocus of −5 μm. Membrane measurements were carried out as previously described [25 (link)]. Briefly, 3dmod from the IMOD package [42 (link)] and custom scripts were used to manually segment the IMs and OMs of projection images of about 35 cells per mutant, measuring the periplasmic width at 0.5-nm intervals to produce width histograms (Fig 2B ).
UltraAuFoil R2/2 grids (200 mesh; Quantifoil Micro Tools GmbH) were glow-discharged for 60 s at 10 mA. Cells were mixed with a solution of 10 nm colloidal gold (Sigma) immediately before freezing. A 2.5-μl droplet of sample was applied to the grid and plunge frozen using a Vitrobot MkIV (FEI Company) with a wait time of 60 s, a blot time of 5 s, a blot force of 3, and a drain time of 1 s at a constant humidity of 100%. Grids were stored under liquid nitrogen until required for data collection.
Projection images were collected on a 200 keV FEI Tecnai TF20 FEG transmission electron microscope (FEI Company) equipped with a Falcon II direct electron detector (FEI Company) using a Gatan 626 cryogenic-holder (Gatan). Leginon automated data-collection software 3.0 [41 (link)] was used to acquire images with pixel size of 0.828 nm (nominal magnification 25,000×) with a defocus of −5 μm. Membrane measurements were carried out as previously described [25 (link)]. Briefly, 3dmod from the IMOD package [42 (link)] and custom scripts were used to manually segment the IMs and OMs of projection images of about 35 cells per mutant, measuring the periplasmic width at 0.5-nm intervals to produce width histograms (
10
Cryo-EM Imaging of Liposome Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3.5 microliters of solution (liposome only, liposome + Xfect, or liposome + rEETI-II) was applied to a freshly glow discharged C-flat holey carbon film with regularly spaced 2 micron holes. The sample was allowed to sit on the grid for 60 seconds then wicked away using Whatman 1 filter paper before additional 3.5 microliters were added to the grid. After 60 seconds incubation, the sample was blotted away for 3.5 seconds and the grids rapidly plunged into liquid ethane using the Vitrobot Mk IV (FEI co.). Grids were then loaded under liquid nitrogen conditions into a Talos F200C (FEI co.) using a Gatan 626 cryo holder (Gatan, Inc). Images were acquired on a CMOS detector (Ceta, FEI co.) at 200 kV using low-dose conditions (~20e/A2/image) at a nominal defocus of ~4 microns.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!