Bm ultrascan

Manufactured by Ametek

Sourced in United States

The BM-Ultrascan is a laboratory equipment product. It is used for ultrasonic scanning and analysis.

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Lab products found in correlation

Falcon 2 camera, by Thermo Fisher Scientific (2 mentions) Tecnai f20 electron microscope, by Thermo Fisher Scientific (1 mentions) Tf20 microscope, by Thermo Fisher Scientific (1 mentions) Tecnai 12 microscope, by Ametek (1 mentions) Em cpc manual plunger, by Leica (1 mentions) Tecnai osiris, by Thermo Fisher Scientific (1 mentions) Filter paper, by Cytiva (1 mentions) Tecnai tf20 tem, by Thermo Fisher Scientific (1 mentions) Vitrobot mark 4, by Thermo Fisher Scientific (1 mentions) Tecnai 12 transmission, by Ametek (1 mentions)

5 protocols using bm ultrascan

Cryo-EM imaging of HIV-1 Env trimer

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HIV-1 Env DS_ConC_SOSIP trimer that contained a disulfide 201C- 433C (DS) was diluted to between 0.0005–0.05 mg/mL in Tris-buffered saline (TBS), pH 7.4, and adhered onto a carbon-coated 200 Cu mesh grid (EMS CF200-Cu) that had been glow discharged in air 2 × 10⁻¹ mbar in air, 25 mA, 30 s, just before use. Subsequently, a 3 μL drop was applied for 1 min followed by blotting with filter paper (Whatman no. 1 or 4). After drying for 1 min, grids were then stained with 3 μL of 2.3% uranyl acetate (UAc) in filtered milliQ (filtered with Millipore filter 0.22 μm) for 60 s, followed by blotting with filter paper (Whatman no. 1 or 4). Data were collected using an FEI Tecnai F20 electron microscope operating at 120 keV, with a magnification of 25,000× that resulted in a pixel size of 4.68 Å at the specimen plane. Images were acquired with a Gatan BM ultrascan.

Rutten L., Lai Y.T., Blokland S., Truan D., Bisschop I.J., Strokappe N.M., Koornneef A., van Manen D., Chuang G.Y., Farney S.K., Schuitemaker H., Kwong P.D, & Langedijk J.P. (2018). A Universal Approach to Optimize the Folding and Stability of Prefusion-Closed HIV-1 Envelope Trimers. Cell reports, 23(2), 584-595.

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Cryo-EM Sample Preparation and Optimization

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For EM grid preparation, the CCNC cryo-EM sample was then dialyzed in 10 mM HEPES, (pH 7.5), 50 mM NaCl, 1 mM EDTA and 1 mM DTT and was diluted with an appropriate volume of the dialysis buffer supplemented with or without NP-40 (0.004%). Negative stain analysis was performed to identify conditions that yielded potentially appropriate concentration, homogeneity, and homogenous distribution of particles for subsequent cryo-EM grid preparations. To do this, 3 µl (0.03 mg/ml) of dialyzed complex was loaded on glow-discharged 300 mesh Cu carbon grids (EMS) and incubated for 30 s before staining with 2% uranyl acetate. All samples were imaged on a FEI Tecnai 12 microscope operating at 120 kV, using a CCD camera (Gatan BM-Ultrascan) at a nominal magnification of 39,000x (Figure S2B). To prepare cryo-EM grids, 2 µl of the dialyzed CCNC (~0.06 and ~0.5 mg/mL without and with 0.004% NP-40) was applied to CF1.2/1.3 holey carbon grids that were glow discharged for 40 s before sample application, subsequently blotted for 8 s, and flash-frozen by plunging into liquid ethane with a Leica EM CPC manual plunger. EM grids were made in batches and freezing conditions were optimized using an FEI TF20 microscope operating at 200 kV equipped with a FEI Falcon II camera.

Allu P.K., Dawicki-McKenna J.M., Eeuwen T.V., Slavin M., Braitbard M., Xu C., Kalisman N., Murakami K, & Black B.E. (2019). Structure of the Human Core Centromeric Nucleosome Complex. Current biology : CB, 29(16), 2625-2639.e5.

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Transmission Electron Microscopy Analysis of Nanoparticles

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TEM analyses were carried out using a TECNAI OSIRIS (Thermo Fisher Scientific, Eindhoven, Netherlands) transmission electron microscope, equipped with a high sensitivity GATAN camera (BM-Ultra-scan, Pleasanton, CA, USA), with 2048 × 2048 pixels. The acceleration voltage was 200 kV using the machine-specific “index 5” for the spot size to reduce possible sample damage induced by the high beam current. An adjustment of the magnification was made to obtain a good pixel number per particle and the number of particles needed for good statistics corresponding to a number of images between 15 and 25. A TIFF image format was used, derived from the GATAN proprietary format (DM4). Here, the method used to determine the dimensional properties of a population of NPs by TEM is based on the thresholding tool developed in the frame of the project by Pollen and included in the Platypus™ software (version specifically developed for nPSize project, 2018, Pollen Metrology, Grenoble, France). At least 100 particles were thresholded and the distributions of the minimum Feret, maximum Feret, and equivalent circle diameters were extracted by using the software.

Feltin N., Crouzier L., Delvallée A., Pellegrino F., Maurino V., Bartczak D., Goenaga-Infante H., Taché O., Marguet S., Testard F., Artous S., Saint-Antonin F., Salzmann C., Deumer J., Gollwitzer C., Koops R., Sebaïhi N., Fontanges R., Neuwirth M., Bergmann D., Hüser D., Klein T, & Hodoroaba V.D. (2023). Metrological Protocols for Reaching Reliable and SI-Traceable Size Results for Multi-Modal and Complexly Shaped Reference Nanoparticles. Nanomaterials, 13(6), 993.

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Negative-Stain EM Analysis of Stabilized Influenza HA

Cited 1 time

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Negative-stain EM was performed on WT H1-CA09, stabilized H1-CA09, WT H3-HK68-folon, stabilized H3-HK68, WT H1-CA09-I53_dn5B, and stabilized H1-CA09-I53_dn5B (NeCen, Leiden University). HA samples were diluted to a concentration of 5 to 20 µg/mL in 20 mM Tris and 150 mM sodium chloride, pH 7.8, and a 4-µL sample was adhered onto a carbon-coated 200-mesh copper grid (Electron Microscopy Sciences) that had been glow discharged (Pelco easiGlow, 25 mA for 45 s) prior to use. The sample drop was applied for 1 min and subsequently blotted with a filter paper (Whatman no. 1 or 4). Following 1-min drying, grids were stained with 3 µL 2% uranyl acetate in filtered Milli-Q (filtered with Millipore filter 0.22 µm) for 60 s and blotted with filter paper (Whatman no. 1 or 4). Data were collected using a Talos L120C electron microscope operating at 120 keV with a magnification of 73,000×. Images were acquired with a Gatan Bm ultrascan. For 2D class-averaged images, 80 to 200 images were collected, and ∼60,000 to 240,000 particles were picked, classified, and averaged using RELION 3.1 (43 (link)). For the WT H3-HK68-foldon and the stabilized H3-HK68 HA with foldon, representative particles were selected and used to generate an ab initio model that was classified in 3D. C3 symmetry was applied to generate the initial model and then relaxed for 3D refinement.

Milder F.J., Jongeneelen M., Ritschel T., Bouchier P., Bisschop I.J., de Man M., Veldman D., Le L., Kaufmann B., Bakkers M.J., Juraszek J., Brandenburg B, & Langedijk J.P. (2022). Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions. Proceedings of the National Academy of Sciences of the United States of America, 119(6), e2115379119.

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Negative Staining and Cryo-EM of Supercomplexes

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For negative staining, purified SCs were diluted to 0.01–0.05 mg/mL concentrations in TBS (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.01% DDM, and 1 mM AEBSF) buffer, 5 μL applied to glow-discharged (20 s, 25 mA, Pelco easiGlow) carbon-coated Cu grids (CF300-CU, EMS), incubated for 1 min, and stained with 2% uranyl acetate. Grids were imaged on FEI Tecnai 12 transmission electron microscope operating at 120 kV, with a CCD camera (Gatan BM-Ultrascan).
For cryo-EM, the tripartite SC was used as purified, whereas the purified bipartite SC was mixed with a 5-fold molar excess of purified cyt c_y^{75 (link)}. In all, 2.5 μL of the mixture, containing 3–5 mg/mL proteins, were applied to CFlat holey carbon grids (1.2/1.3–400 mesh or 2/2–300 mesh) (EMS), which were glow discharged (2 min, 25 mA, Pelco easiGlow) before sample application. Grids were blotted for 9 s at force 0 (CFlat-1.2/1.3) or for 3 s at force −5 (CFlat-2/2) and flash-frozen in liquid ethane cooled with liquid nitrogen using FEI Vitrobot Mark IV (25 °C, 100% humidity). Plunge freezing conditions were optimized using FEI Tecnai TF20 TEM operating at 200 kV, equipped with an FEI Falcon II camera.

Steimle S., van Eeuwen T., Ozturk Y., Kim H.J., Braitbard M., Selamoglu N., Garcia B.A., Schneidman-Duhovny D., Murakami K, & Daldal F. (2021). Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes. Nature Communications, 12, 929.

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