Em gp automatic plunge freezer

Manufactured by Leica

Sourced in Germany

The EM-GP automatic plunge Freezer is a lab equipment designed for rapid freezing of biological samples. It provides a controlled environment for the freezing process, ensuring consistent and reproducible results. The core function of this device is to quickly freeze samples in a manner that preserves their structural integrity, making it suitable for various applications in the field of electron microscopy and related research areas.

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Lab products found in correlation

Whatman, by Cytiva (4 mentions) Ultraufoil r1.2 1 3 300 mesh grid, by Quantifoil (4 mentions) Atpγs, by Merck Group (2 mentions) Superose 6 10 300 gl column, by GE Healthcare (2 mentions) Falcon 3 direct electron detector, by Thermo Fisher Scientific (1 mentions) Epu software, by Thermo Fisher Scientific (1 mentions) Titan krios tem, by Thermo Fisher Scientific (1 mentions) Copper grid, by Ted Pella (1 mentions) Jem 2200f, by JEOL (1 mentions) Tecnai arctica microscope, by Thermo Fisher Scientific (1 mentions) R3.5 1, by Quantifoil (1 mentions) N 1 filter paper, by Cytiva (1 mentions) Gif quantum energy filter, by Ametek (1 mentions) Us4000 ccd camera, by Ametek (1 mentions) 626 cryo holder, by Ametek (1 mentions) Gold nanoparticles, by BBI Solutions (1 mentions) Fesem merlin, by Zeiss (1 mentions) Silicon wafer, by Ted Pella (1 mentions) Cryo transfer specimen holder, by Ametek (1 mentions) Whatman filter paper number 1, by GE Healthcare (1 mentions)

Close spelling variants of this product

EM GP (197 citations) EM GP plunge freezer (32 citations) Automatic plunge freezer (7 citations) GP plunge freezer (3 citations)

13 protocols using em gp automatic plunge freezer

Structural Analysis of RuBisCO and α-Carboxysomes

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For the structural characterisation of RuBisCO, 3 µL aliquots of purified α-carboxysomes at a concentration of ~1 mg mL -1 were applied to Graphene Oxide coated, 300 mesh, 2/2 µm hole/spacing, holey carbon grids (EMR). A Leica EM GP Automatic Plunge Freezer (Leica) was used to plunge freeze the sample, blotting for 3-6 s. Cryo-EM data was collected with a 300 kV Titan Krios TEM, equipped with a Falcon 3 direct electron detector (Thermo Fisher) operated in linear mode. 4593 micrographs were collected using the EPU software (Thermo Fisher) with a pixel size of 1.11 Å pix -1 , a total dose rate of 30 e -Å -2 , and 44 fractions per micrograph. The defocus range was -0.5 to -1.5 µm.
For structural characterisation of the intact α-carboxysome complex, 3 µL aliquots of purified sample at a concentration of 3 mg mL -1 were applied to Graphene Oxide coated grids, 300 mesh, 2/2 µm hole/spacing, holey carbon grids (EMR). A Leica EM GP Automatic Plunge Freezer (Leica) was used to plunge freeze, blotting for 6 s. Cryo-EM data were collected with a 300 kV Titan Krios TEM with a Falcon 3 direct electron detector (FEI) operated in counting mode. 5429 micrographs were collected using EPU software (Thermo Fisher) with a pixel size of 2.23 Å pix -1 with a total dose rate of 29.7 e -Å -2 with 33 frames per micrograph. The defocus range was -1.0 to -2.2 µm.

Evans S.L., Al-Hazeem M.M.J., Mann D., Smetacek N., Beavil A.J., Sun Y., Chen T., Dykes G.F., Liu L., & Bergeron J.R.C. (2022). Single-particle cryo-EM analysis of the shell architecture and internal organization of an intact α-carboxysome.

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Cryogenic Transmission Electron Microscopy

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Samples were gently dispersed by pipetting up and down, and small aliquots of each sample (4 μL) were transferred to a glow-discharged lacey carbon film on a copper grid (Ted Pella, Redding, CA). The grid with the sample was then quickly frozen in liquid ethane using a Leica EM GP automatic plunge freezer (Leica, Wetzlar, Germany). Samples were stored in liquid nitrogen and transferred into the microscope using a Fischione Model 2550 Cryo Transfer Tomography Holder (E.A. Fischione Instruments, Export, PA). The samples were then imaged using a JEOL (Tokyo, Japan) JEM-2200FS transmission electron microscope equipped with an in-column omega energy filter at 200 kV accelerating voltage. Images were captured digitally using a Tietz Video and Image Processing Systems (Gauting, Germany) TemCam-F416 camera.

Pallbo J., Olsson U, & Sparr E. (2021). Strong inhibition of peptide amyloid formation by a fatty acid. Biophysical Journal, 120(20), 4536-4546.

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Cryo-TEM of Pickering Nanoemulsions

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Imaging was performed using
an FEI Tecnai Arctica microscope operating
at an acceleration voltage of 200 kV. Cryo-TEM samples were prepared
by depositing 5 μL of a 0.5% w/w dodecane-in-water or squalane-in-water
Pickering nanoemulsion onto a plasma-treated Quantifoil holey carbon-coated
copper grid, followed by blotting for approximately 4 s and then plunging
into a pool of liquid ethane to vitrify the sample using a Leica EM
GP automatic plunge freezer (25 °C, 99% humidity). Transfer of
the vitrified grids into a precooled cryo-TEM holder was performed
at −196 °C prior to microscopic analysis.

Hunter S.J., Chohan P., Varlas S, & Armes S.P. (2024). Effect of Temperature, Oil Type, and Copolymer Concentration on the Long-Term Stability of Oil-in-Water Pickering Nanoemulsions Prepared Using Diblock Copolymer Nanoparticles. Langmuir, 40(7), 3702-3714.

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Negative Staining and Cryo-EM Sample Preparation

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For negative stain analysis the cross-linked sample was diluted in size-exclusion buffer to around 0.2 mg/mL. A 3.5 μL aliquot of the sample was loaded onto a freshly glow discharged thin carbon-coated grid (Quantifoil R3.5/1 with a 2 nm carbon coating), incubated for 45 s at room temperature and washed with six drops of size-exclusion buffer. The grid was then stained with a drop of 2% uranyl-acetate for 15 s and dried on a filter paper.
For cryo-EM grids, the cross-linked complex was diluted to around 0.4 mg/ml. Four microliters of the sample were loaded on a previously glow discharged holey carbon (Quantifoil R2/1) grid. After 1 s of blotting time, grids were plunge frozen in liquid ethane by using a Leica EM GP-Automatic Plunge Freezer. The temperature in the chamber was kept at +15°C and the humidity at 95%.

Ivic N., Potocnjak M., Solis-Mezarino V., Herzog F., Bilokapic S, & Halic M. (2019). Fuzzy Interactions Form and Shape the Histone Transport Complex. Molecular Cell, 73(6), 1191-1203.e6.

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Cryo-EM Analysis of Extracellular Vesicle Morphology

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To analyze the morphology of EVs by cryo-EM, 3 µL of EV sample was first deposited onto a glow-discharged 200-mesh lacey carbon grid. Prior to freezing, the grid was loaded into the thermostatic chamber of a Leica EM-GP automatic plunge Freezer, set to 20°C and 95% humidity. Excess solution was blotted from the grid for 1–2 s with a Whatman n°1 filter paper and the grid immediately flash frozen in liquid ethane cooled to −185°C. Specimens were then transferred into a Gatan 626 cryo-holder. Cryo EM was carried out using a Jeol 2,100 microscope equipped with a LaB6 cathode operating at 200 kV under low-dose conditions. Images were acquired using SerialEM software (Mastronarde, 2005 (link)), with the defocus ranging from 600 to 1,000 nm, using a Gatan US4000 CCD camera. This device was placed at the end of a GIF quantum energy filter (Gatan Inc. Berwyn, Pennsylvania, United States) operating in zero-energy-loss mode, with a slit width of 25 eV. Images were recorded at a magnification corresponding to a calibrated pixel size of 0.87 Å.

Yaker L., Tebani A., Lesueur C., Dias C., Jung V., Bekri S., Guerrera I.C., Kamel S., Ausseil J, & Boullier A. (2022). Extracellular Vesicles From LPS-Treated Macrophages Aggravate Smooth Muscle Cell Calcification by Propagating Inflammation and Oxidative Stress. Frontiers in Cell and Developmental Biology, 10, 823450.

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Cryogenic Imaging of Live U2OS Cells

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Human osteosarcoma U2OS cells expressing LifeAct-mRFPruby (Riedl et al., 2008 ▸ ) were a kind gift from E. Manser and Y. Baskaran (A*STAR, Singapore). Cells were grown adherent at 37°C in 5% fetal bovine serum (FBS) in complete Dulbeccos’ modified Eagle’s medium (DMEM) supplemented with penicillin/streptomycin. Cells were lifted from the plates using trypsin/ethylenediaminetetraacetic acid (EDTA) and were used to seed 3 mm carbon-coated EM grids (Quantifoil) in the same medium. Once they had adhered, they were allowed to grow to 60–70% confluence, whereupon the grids were removed from the medium, fiducialized using 250 nm diameter gold nanoparticles (BBI Solutions), gently blotted and vitrified by plunge-freezing into liquid nitrogen-cooled liquid ethane using an EM GP Automatic Plunge Freezer (Leica). The cryopreservation process immobilizes the sample in native conditions and provides resistance to radiation damage during imaging. Samples were thereafter stored in liquid nitrogen until retrieved for imaging. All samples were prepared following established protocols as documented previously (Okolo et al., 2021 ▸ ).

Koronfel M., Kounatidis I., Mwangangi D.M., Vyas N., Okolo C., Jadhav A., Fish T., Chotchuang P., Schulte A., Robinson R.C, & Harkiolaki M. (2021). Correlative cryo-imaging of the cellular universe with soft X-rays and laser light used to track F-actin structures in mammalian cells. Acta Crystallographica. Section D, Structural Biology, 77(Pt 12), 1479-1485.

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Nanoparticle Morphometry by Electron Microscopy

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Ultrastructural characterization of nanoparticle morphometry (size and shape) was determined at nearly native state with two electron microscopy techniques using cryoTEM and FESEM. In a qualitative approach with cryoTEM, microdrops of T22-BAK-GFP-H6, T22-PUMA-GFP-H6, and T22-BAXPORO-GFP-H6 at 0.3 mg/ml in their respective storage buffers were deposited in Holey carbon-coated copper grids (400 mesh), cryofixed in liquid ethane with an EM GP automatic plunge freezer (Leica), placed in a cryo-transfer specimen holder (Gatan Inc.) and observed in a TEM JEM-2011 (Jeol) operating at 200 kV and equipped with a 895 USC 4000 CCD camera (Gatan Inc.). Representative images of general fields and nanoparticle details were captured at high magnifications (from 40,000x to 150,000x). In qualitative and quantitative approaches with FESEM, drops of the same three samples at the same conditions were directly deposited on silicon wafers (Ted Pella Inc.) for one minute.
The excess of liquid was blotted with Whatman filter paper number 1 (GE Healthcare), and samples were air dried for a few minutes and immediately observed without coating with a FESEM Merlin (Zeiss) operating at 1 kV and equipped with a high resolution in-lens secondary electron detector. Representative images of general fields and nanoparticle details were captured at high magnifications (from 100,000x to 400,000x).

Sánchez-García L., Sala R., Serna N., Álamo P., Parladé E., Alba-Castellón L., Voltà-Durán E., Sánchez-Chardi A., Unzueta U., Vázquez E., Mangues R, & Villaverde A. (2020). A refined cocktailing of pro-apoptotic nanoparticles boosts anti-tumor activity. Acta biomaterialia, 113.

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Cryo-EM Grid Preparation for Cell Vitrification

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Gold EM grids with Quantifoil R 1.2/20 or R 1/4 holey carbon film (or R 1/4 holey S_iO₂ film for the cytochalasin D data; Quantifoil Micro Tools GmbH) were glow-discharged in an EasiGlow (Pelco) glow discharge system. After grid sterilization under UV light, the cell suspension containing fiducials was seeded onto the grids and incubated for 2 h at 37 °C to let the cells adhere to the grids, resulting in 3 to 4 cells per grid square. For cell vitrification, grids were loaded into the thermostatic chamber of a Leica EM-GP automatic plunge freezer, set at 20 °C and 95% humidity. Excess solution was blotted away for 10 s with a Whatman filter paper no. 1, and the grids were immediately flash frozen in liquid ethane cooled at −185 °C.

Jasnin M., Hervy J., Balor S., Bouissou A., Proag A., Voituriez R., Schneider J., Mangeat T., Maridonneau-Parini I., Baumeister W., Dmitrieff S, & Poincloux R. (2022). Elasticity of podosome actin networks produces nanonewton protrusive forces. Nature Communications, 13, 3842.

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Cryo-EM Sample Preparation and Imaging

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For cryo-EM, 3.5 μl of sample (at a final RNA concentration of ∼20 ng/μl as estimated by Nanodrop measurements), were deposited onto freshly glow-discharged holey carbon grids (Quantifoil R2/1 or R2/2) and loaded into the thermostatic chamber of a Leica EM-GP automatic plunge Freezer, set at 20°C and 95% humidity. Excess solution was blotted for 1.8–2.1 s with a Whatman filter paper no. 1, and the grid was immediately flash frozen in liquid ethane, cooled at -183°C. Grids were prepared and systematically checked at METI, the Toulouse cryo-EM facility. Cryo-EM image acquisition was performed at the Netherlands Center for Electron Nanoscopy (NeCEN), using a FEI Titan Krios Microscope operating at 300 kV. Using the FEI EPU software, images were automatically recorded on a Falcon II direct electron detector, with an electron dose of ∼30 e⁻/Å², at a nominal magnification of 59 000× corresponding to a calibrated pixel size of 1.136 Å/pixel. Nominal defoci ranged from −1.6 to −2.5 μm.

Larburu N., Montellese C., O'Donohue M.F., Kutay U., Gleizes P.E, & Plisson-Chastang C. (2016). Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing. Nucleic Acids Research, 44(17), 8465-8478.

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Structural Analysis of CtLas1-Grc3 Complex

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Purified CtLas1-Grc3 complex (10 μM) was cross-linked with 50 μM bis(sulfosuccinimidyl)suberate (BS3; Sigma) in 20 mM Hepes pH 7.7, 200 mM NaCl, 5 mM MgCl₂, 5% glycerol at room temperature for 5 minutes before quenching with 30 mM Tris pH 7.5 for 15 minutes at 4°C. Cross-linked reactions were resolved over a Superose 6 10/300 GL column (GE Healthcare) in 10 mM Tris pH 8.0, 200 mM NaCl, 5 mM MgCl₂. CtLas1-Grc3 (0.15 mg/ml) was incubated in the absence (apo state) and presence (ATP-γS bound state) of 2 mM ATP-γS (Sigma) and 10 μM CT-ITS2-RNA (5′-UGUGUUGGGGdeoxyACCCGCGGCUGCUCG CGGGCCCUGAAAAGCA-3′) for 1 hour at 4°C. The CT-ITS2-RNA substrate represents part of the C. thermophilum ITS2 pre-rRNA sequence. Protein mixtures (3 μL) were applied to glow-discharged UltrAuFoil R1.2/1.3 300 mesh grids (Quantifoil) and vitrified after 5 sec blotting using the Automatic Plunge Freezer EM GP (Leica).

Pillon M.C., Hsu A.L., Krahn J.M., Williams J.G., Goslen K.H., Sobhany M., Borgnia M.J, & Stanley R.E. (2019). Cryo-EM Reveals Active Site Coordination Within a Multienzyme pre-rRNA Processing Complex. Nature structural & molecular biology, 26(9), 830-839.

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