5. Cross-linkers. Chemical cross-linkers such as glutaraldehyde
(Sigma, ref.: G5882) or bis-sulfosuccinimidyl suberate (BS3,
Thermo Fisher Scientific, ref.: 21580) are often used to stabilize fragile sample by forming chemical bonds between proximal lysine residues. Chemical cross-linkers have a short life time
and may form oligomeric states. They can be aliquoted and
frozen to extend their life time.
2.2 Negative
Staining
1. Heavy atom salt solution. Uranyl acetate (Agar Scientific, ref.:
AGR1260A; Polysciences ref.: 21447-25) uranyl formate,
phosphotungstic acid (PTA), and other heavy metals are commonly used to embed the biological sample into a shell of
electron-dense material. The heavy atom salt is dissolved in
water at 2% (w/v). Most of the solutions are at low pH, and
only few of them, such as PTA, can be buffered to pH 7.0
without forming aggregates. In this respect, phosphate buffer
present in the sample solution may provoke uranyl acetate
precipitation observed as dark clusters under the microscope.
2. Handling heavy metal salts requires strict safety protections:
use gloves, mask and safety glasses and work under hood when
preparing solutions. Uranyl salts are slightly radioactive, and
specific safety precautions are necessary: store uranyl salts in a
lead protection box, identify the workbench used to work with
uranyl acetate, weigh powders in a secured work station, work
under a hood protected by a Plexiglas screen when handling
concentrated solutions, and finally, make sure to dispose any
contaminated waste according to your lab policy. Radiations
emitted from 2% solutions are shielded by water molecules and
the glass bottle. Depending on national policies, uranyl-based
reagent might be difficult or even illegal to buy. Please make
sure your experiments comply with your local regulations.
3. Note that there are now non-radioactive alternatives to uranyl
acetate: based on methylamine vanadate (NanoVan, Nanoprobes), methylamine tungstate (Nano-W, Nanoprobes), UranyLess (TAAB), and UA-Zero (Agar Scientific).
4. Parafilm. The protocol includes several washing steps aiming at
solution replacement. Parafilm may be used to deposit small
solution drops on top of which the EM grid can be placed
(VWR international SAS, ref.: 97949).
2.3 Cryo-EM
Preparing the sample for cryo-EM requires more material and
equipment than negative staining. Although automated spray systems and microfluidic-based devices are emerging, vitrification is
usually performed using a cryo-plunger. Make sure that all the
materials are at your disposal before you start since the protocol
cannot be interrupted.
Specimen Optimization for Single Particle cryo-EM
247
(Sigma, ref.: G5882) or bis-sulfosuccinimidyl suberate (BS3,
Thermo Fisher Scientific, ref.: 21580) are often used to stabilize fragile sample by forming chemical bonds between proximal lysine residues. Chemical cross-linkers have a short life time
and may form oligomeric states. They can be aliquoted and
frozen to extend their life time.
2.2 Negative
Staining
1. Heavy atom salt solution. Uranyl acetate (Agar Scientific, ref.:
AGR1260A; Polysciences ref.: 21447-25) uranyl formate,
phosphotungstic acid (PTA), and other heavy metals are commonly used to embed the biological sample into a shell of
electron-dense material. The heavy atom salt is dissolved in
water at 2% (w/v). Most of the solutions are at low pH, and
only few of them, such as PTA, can be buffered to pH 7.0
without forming aggregates. In this respect, phosphate buffer
present in the sample solution may provoke uranyl acetate
precipitation observed as dark clusters under the microscope.
2. Handling heavy metal salts requires strict safety protections:
use gloves, mask and safety glasses and work under hood when
preparing solutions. Uranyl salts are slightly radioactive, and
specific safety precautions are necessary: store uranyl salts in a
lead protection box, identify the workbench used to work with
uranyl acetate, weigh powders in a secured work station, work
under a hood protected by a Plexiglas screen when handling
concentrated solutions, and finally, make sure to dispose any
contaminated waste according to your lab policy. Radiations
emitted from 2% solutions are shielded by water molecules and
the glass bottle. Depending on national policies, uranyl-based
reagent might be difficult or even illegal to buy. Please make
sure your experiments comply with your local regulations.
3. Note that there are now non-radioactive alternatives to uranyl
acetate: based on methylamine vanadate (NanoVan, Nanoprobes), methylamine tungstate (Nano-W, Nanoprobes), UranyLess (TAAB), and UA-Zero (Agar Scientific).
4. Parafilm. The protocol includes several washing steps aiming at
solution replacement. Parafilm may be used to deposit small
solution drops on top of which the EM grid can be placed
(VWR international SAS, ref.: 97949).
2.3 Cryo-EM
Preparing the sample for cryo-EM requires more material and
equipment than negative staining. Although automated spray systems and microfluidic-based devices are emerging, vitrification is
usually performed using a cryo-plunger. Make sure that all the
materials are at your disposal before you start since the protocol
cannot be interrupted.
Specimen Optimization for Single Particle cryo-EM
247