The peak fraction corresponding to the Hsp104DWB hexamer was incubated at 22°C for 10 min with 5 mM ATP in buffer consisting of 50 mM MOPS pH 7.5, 2 mM DTT, and 10 mM MgCl2, and kept on ice for another 30 min. 3.0 μl of sample (0.3 mg/ml) was applied to glow discharged copper grids (Quantifoil, Groβlöbichau, Germany), blotted, and frozen in liquid ethane. Frozen hydrated grids were imaged using a 300-kV Polara G2 electron microscope (FEI, Hillsboro, OR) equipped with a field emission gun. Images were recorded on an electron counting K2 Summit direct detection camera (Gatan Inc., Pleasanton, CA) operated in super-resolution mode at 23,000× nominal magnification corresponding to 1.68 Å/pixel. Dose fractionated imaging was performed by semi-automated collection methods using SerialEM (Mastronarde, 2005 (link)). 7.6 s exposures were collected at 200 msec/frame, with a total electron dose of 38 e− per micrograph in 38 frames.
Glow discharged copper grids
Manufactured by Quantifoil
Sourced in Germany
Glow discharged copper grids are laboratory equipment used for sample preparation in electron microscopy. They provide a uniform surface charge to facilitate the adhesion of biological samples, such as proteins or cells, onto the grid for subsequent imaging and analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using glow discharged copper grids
1
Cryo-EM Imaging of Hsp104 Hexamer
2
Cryo-EM Imaging of Hsp104 Hexamer
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The peak fraction corresponding to the Hsp104DWB hexamer was incubated at 22°C for 10 min with 5 mM ATP in buffer consisting of 50 mM MOPS pH 7.5, 2 mM DTT, and 10 mM MgCl2, and kept on ice for another 30 min. 3.0 μl of sample (0.3 mg/ml) was applied to glow discharged copper grids (Quantifoil, Groβlöbichau, Germany), blotted, and frozen in liquid ethane. Frozen hydrated grids were imaged using a 300-kV Polara G2 electron microscope (FEI, Hillsboro, OR) equipped with a field emission gun. Images were recorded on an electron counting K2 Summit direct detection camera (Gatan Inc., Pleasanton, CA) operated in super-resolution mode at 23,000× nominal magnification corresponding to 1.68 Å/pixel. Dose fractionated imaging was performed by semi-automated collection methods using SerialEM (Mastronarde, 2005 (link)). 7.6 s exposures were collected at 200 msec/frame, with a total electron dose of 38 e− per micrograph in 38 frames.
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