After 24 hours of growth, the tube of V. cholerae culture was placed on a bench at room temperature for ~1 min to sediment visible microcolonies. After the pellet was formed the supernatant was gently removed by pipetting and the pellet was resuspended in fresh LB. The cells were incubated for 15 minutes prior to mixing with 10-nm colloidal gold (Sigma-Aldrich, St. Louis, MO) pretreated with bovine serum albumin and subsequently gently applied to freshly glow-discharged Quantifoil copper R2/2 200 EM grid (Quantifoil Micro Tools GmbH, Jena, Germany). The grids were plunge-frozen in a liquid ethane propane mixture50 using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Grids were stored in liquid nitrogen prior to imaging.
10 nm colloidal gold
Manufactured by Merck Group
Sourced in Germany, United States
10-nm colloidal gold is a laboratory product consisting of spherical gold nanoparticles with an average diameter of 10 nanometers, dispersed in an aqueous solution. The primary function of this product is to serve as a contrast agent or labeling material in various scientific applications, such as electron microscopy, immunoassays, and biological imaging.
Automatically generated - may contain errors
Lab products found in correlation
Copper r2 2 200 em grid, by Quantifoil (2 mentions)
Fei vitrobot mark 4, by Thermo Fisher Scientific (2 mentions)
Holey carbon grid, by Quantifoil (1 mentions)
Tecnai f30 tem, by Thermo Fisher Scientific (1 mentions)
Cryo holder, by Ametek (1 mentions)
Fei vitrobot mark 3, by Thermo Fisher Scientific (1 mentions)
6 protocols using 10 nm colloidal gold
1
Cryo-EM Sample Preparation for V. cholerae
2
Rat Anti-VIT-2 Antibody Protocol
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The rat polyclonal anti‐VIT‐2 antibody (diluted 1:100 for immuno‐EM labeling) was kindly provided by Dr. Xiao‐Chen Wang (Institute of Biophysics, Chinese Academy of Sciences, Beijing, China) (Liu et al., 2012 (link)). The epitope of the antibody is a recombinant protein VIT‐2 (83–620 amino acid)::6xHIS. The rabbit‐derived second antibody (anti‐rat) conjugated with 10‐nm colloidal gold (Sigma) is available as a commercial product.
3
Cryo-EM Sample Preparation for V. cholerae
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After 24 hours of growth, the tube of V. cholerae culture was placed on a bench at room temperature for ~1 min to sediment visible microcolonies. After the pellet was formed the supernatant was gently removed by pipetting and the pellet was resuspended in fresh LB. The cells were incubated for 15 minutes prior to mixing with 10-nm colloidal gold (Sigma-Aldrich, St. Louis, MO) pretreated with bovine serum albumin and subsequently gently applied to freshly glow-discharged Quantifoil copper R2/2 200 EM grid (Quantifoil Micro Tools GmbH, Jena, Germany). The grids were plunge-frozen in a liquid ethane propane mixture50 using an FEI Vitrobot Mark IV (FEI Company, Hillsboro, OR). Grids were stored in liquid nitrogen prior to imaging.
4
Cryogenic Transmission Electron Microscopy of Axonemes
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Quantifoil holey carbon grids (Quantifoil Micro Tools, Jena, Germany) were glow discharged and then coated with 10-nm colloidal gold (Sigma-Aldrich). After loading the grid in a home-made plunge freezer, 3 μl of axonemes and 1 μl of a 5× concentrated, 10-nm colloidal gold solution were applied to the grid. The grid was blotted for ∼2 s with a filter paper and then immediately plunge frozen in liquid ethane to achieve vitrification. The frozen samples were stored in liquid nitrogen until TEM examination.
Using a cryo-holder (Gatan, Pleasanton, CA) to maintain the sample at a temperature below −140°C, a vitrified sample was transferred into a Tecnai F30 TEM (FEI, Eindhoven, Netherlands) equipped with a field emission gun and a postcolumn energy filter (Gatan). Images were recorded at 300 keV under low-dose conditions and in the zero-loss mode of the energy filter (20-eV slit width). Tilt series of axoneme samples were automatically recorded from −65 to +65º with 1.5–2.5° angular increments using SerialEM software (Mastronarde, 2005 (link)). The cumulative electron dose was restricted to ∼100 e/Å2. All data were acquired with a 2k × 2k charge-coupled device camera (Gatan) at −8-μm defocus and at a magnification of 13,500×, resulting in a pixel size of ∼1 nm.
Using a cryo-holder (Gatan, Pleasanton, CA) to maintain the sample at a temperature below −140°C, a vitrified sample was transferred into a Tecnai F30 TEM (FEI, Eindhoven, Netherlands) equipped with a field emission gun and a postcolumn energy filter (Gatan). Images were recorded at 300 keV under low-dose conditions and in the zero-loss mode of the energy filter (20-eV slit width). Tilt series of axoneme samples were automatically recorded from −65 to +65º with 1.5–2.5° angular increments using SerialEM software (Mastronarde, 2005 (link)). The cumulative electron dose was restricted to ∼100 e/Å2. All data were acquired with a 2k × 2k charge-coupled device camera (Gatan) at −8-μm defocus and at a magnification of 13,500×, resulting in a pixel size of ∼1 nm.
5
Cryo-Electron Tomography of Bacterial Cells
6
Cryo-EM Sample Preparation for C. jejuni
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Sample preparation closely followed established procedures (Iancu et al. 2007 (link)). In brief, 4 μL of a 10 nm colloidal gold (Sigma Aldrich, St. Louis, MO, USA) in 5% Bovine serum albumin (BSA) was added to 16 μL of a C. jejuni culture that had been allowed to grow to an optical density of 0.5. Three microliters of this mix were then placed onto a glow discharged carbon-coated R 2/2 Quantifoil grid in a Vitrobot (FEI Company, Hillsboro, OR). Prior to this a 10 nm colloidal gold suspension in 5% BSA solution was added to the Quantifoil grid and allowed to dry. The temperature in the Vitrobot™ chamber was kept at 22°C with 100% humidity. Placing the sample onto the grid was followed by a 1 sec blot with an offset of −1.5°, a drain time of 1 sec, and plunge frozen in a mixture of liquid ethane (63%) and propane (37%). The frozen grids were than stored in liquid nitrogen until further use.
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