Purified protein samples were first diluted to a concentration of approximately 0.1 mg/ml using a buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM DTT, and subsequently diluted in a 1:1 ratio in the spraying buffer containing 200 mM ammonium acetate and 60% (v/v) glycerol, pH 7.6. After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with a 0.7-nm-thick layer of platinum (BALTIC, Germany) at an angle of 4–5°, followed by a 6–7 nm layer of carbon (Balzers, Liechtenstein) at 90°. The replicas obtained were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and observed in an FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
Mica chips
Manufactured by Agar Scientific
Sourced in United Kingdom
Mica chips are thin, transparent sheets of the mineral mica. They are used in various laboratory applications due to their unique physical and electrical properties.
Automatically generated - may contain errors
4 protocols using mica chips
1
Protein Sample Preparation for TEM Imaging
2
Cohesin Complexes Analyzed by EM
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Cohesin was diluted to a concentration of approximately 0.1 mg/mL in FLAG buffer in the absence or presence of 1 μM rapamycin. Hinge domains were diluted to 0.1 mg/ml in 20 mM Tris pH 7.5, 100 mM NaCl. Samples were subsequently diluted 1:1 in spraying buffer, containing 200 mM ammonium acetate and 60% (v/v) glycerol, pH adjusted to 7.6.
After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 8 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, the Netherlands) operated at 80 kV. Positions on the grid were chosen randomly. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 8 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, the Netherlands) operated at 80 kV. Positions on the grid were chosen randomly. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
3
Cohesin Trimer Preparation and Imaging
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Cohesin trimer was first diluted to a concentration of approximately 0.1 mg mL−1 in 50 mM sodium phosphate buffer pH 7.6 (including 150 mM NaCl, 5% glycerol and 0.5 mM TCEP) and subsequently diluted 1:1 in spraying buffer, containing 200 mM ammonium acetate and 60% (v/v) glycerol, pH adjusted to 7.6. After dilution, the samples were sprayed onto freshly cleaved mica chips (Agar Scientific, UK) and immediately transferred into a BAL-TEC MED020 high vacuum evaporator (BAL-TEC, Liechtenstein) equipped with electron guns. While rotating, samples were coated with 0.7 nm Platinum (BALTIC, Germany) at an angle of 4-5°, followed by 7 nm Carbon (Balzers, Liechtenstein) at 90°. The obtained replicas were floated off from the mica chips, picked up on 400 mesh Cu/Pd grids (Agar Scientific, UK), and inspected in an FEI Morgagni 268D TEM (FEI, The Netherlands) operated at 80 kV. Images were acquired using an 11 megapixel Morada CCD camera (Olympus-SIS, Germany).
4
Platinum-carbon coating for RNA imaging
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Ex-virion RNA in 100 mM Na+ or K+ phosphate buffer (pH 7.4) was prepared as detailed in Section 2.4 . The RNA samples were first diluted to ~0.1 mg/mL in the absence (control) or presence of 20 µM PDS and incubated for 10 min at ambient temperature. The samples were subsequently diluted 1:1 in spraying buffer (200 mM ammonium acetate and 60% (v/v) glycerol, pH adjusted to 7.4). Samples were immediately sprayed onto freshly cleaved mica chips (Agar Scientific, Birchanger, Essex, UK) and quickly transferred into a BAL-TEC MED020 high vacuum evaporator equipped with electron guns. While rotating, samples were coated with 0.6 nm platinum (BALTIC) at an angle of 7°, followed by 6 nm carbon (Balzers, Liechtenstein) at 90°. The replicas were floated off the mica chips, picked up on 400-mesh Cu/Pd grids (Agar Scientific), and inspected in an FEI Morgagni 268D TEM (Thermo Fisher Scientific) operated at 80 kV. Images were acquired using an 11-megapixel Morada CCD camera (Olympus SIS, Münster, Germany).
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