evaporation, a force- and time-controlled blotting and automated plunging [10] (Vitrobot Mark IV, Thermo Fisher; EM
GP2, Leica).
8. Ethane gas cylinder. A 5 L cylinder containing compressed
ethane gas is required to fill the ethane container. The cylinder
has to be equipped with a pressure reducer to fine tune the gas
flow and with a thin flexible plastic tube (diameter of 2 mm) to
flow the ethane stream into the liquid nitrogen-cooled cryogen
container. The extremity of a pipette tip can be mounted at the
end of the plastic tube for increased precision in driving the
ethane flow.
9. Safety issues. Handling liquid nitrogen and ethane which are
very cold and vaporize into large amounts of gas requires strict
safety protections. Liquid ethane close to its solidification temperature is dangerous and will freezes any hydrated object upon
contact. Care must be taken to avoid spills on skin or eyes. The
use of a face shield is highly recommended. Thick gloves must
be used to manipulate cold objects.
3 Methods
3.1 Negative
Staining
Negative staining is a fast method to assess the quality of your
sample by checking its stability, homogeneity, and propensity to
aggregate. Key steps of sample preparation are illustrated in Fig. 1
and detailed below.
3.1.1 Preparation of a
Negatively Stained Sample
1. Glow discharge a carbon-coated EM grid to render the carbon
support hydrophilic. Place the EM grid in the glow discharging
device, carbon side facing up. The nature of the gas present in
the chamber, the partial gas pressure, the voltage applied, the
glow discharge time, and the geometry of the device will all
affect the ionization of the carbon support. Using air as the
residual gas at a pressure of 1.8 Â 10
À1 bar, a current of 2 mA
should be applied for 45 s to assure a suitable hydrophilicity
(Fig. 1a).
2. Deposit 3–5 μL of your sample onto the glow discharged side
of the grid held with tweezers and wait for 1 min for protein
adsorption. The protein concentration should be around
30 μg/mL (Fig. 1b).
3. Place a square of parafilm onto the bench and deposit four
separate 40 μL drops, three drops of water or buffer, and one
drop of phosphotungstic acid at 2%.
4. After adsorption (step 2), place the grid sequentially on top of
the first three droplets of water to remove the material not
adsorbed to the carbon (Fig. 1c). Then remove the excess of
Specimen Optimization for Single Particle cryo-EM
249
GP2, Leica).
8. Ethane gas cylinder. A 5 L cylinder containing compressed
ethane gas is required to fill the ethane container. The cylinder
has to be equipped with a pressure reducer to fine tune the gas
flow and with a thin flexible plastic tube (diameter of 2 mm) to
flow the ethane stream into the liquid nitrogen-cooled cryogen
container. The extremity of a pipette tip can be mounted at the
end of the plastic tube for increased precision in driving the
ethane flow.
9. Safety issues. Handling liquid nitrogen and ethane which are
very cold and vaporize into large amounts of gas requires strict
safety protections. Liquid ethane close to its solidification temperature is dangerous and will freezes any hydrated object upon
contact. Care must be taken to avoid spills on skin or eyes. The
use of a face shield is highly recommended. Thick gloves must
be used to manipulate cold objects.
3 Methods
3.1 Negative
Staining
Negative staining is a fast method to assess the quality of your
sample by checking its stability, homogeneity, and propensity to
aggregate. Key steps of sample preparation are illustrated in Fig. 1
and detailed below.
3.1.1 Preparation of a
Negatively Stained Sample
1. Glow discharge a carbon-coated EM grid to render the carbon
support hydrophilic. Place the EM grid in the glow discharging
device, carbon side facing up. The nature of the gas present in
the chamber, the partial gas pressure, the voltage applied, the
glow discharge time, and the geometry of the device will all
affect the ionization of the carbon support. Using air as the
residual gas at a pressure of 1.8 Â 10
À1 bar, a current of 2 mA
should be applied for 45 s to assure a suitable hydrophilicity
(Fig. 1a).
2. Deposit 3–5 μL of your sample onto the glow discharged side
of the grid held with tweezers and wait for 1 min for protein
adsorption. The protein concentration should be around
30 μg/mL (Fig. 1b).
3. Place a square of parafilm onto the bench and deposit four
separate 40 μL drops, three drops of water or buffer, and one
drop of phosphotungstic acid at 2%.
4. After adsorption (step 2), place the grid sequentially on top of
the first three droplets of water to remove the material not
adsorbed to the carbon (Fig. 1c). Then remove the excess of
Specimen Optimization for Single Particle cryo-EM
249