applications, it may be advantageous to coat grids on a per user
basis—detailed protocols are available [15]. Coating grids
allows the user to create thin carbon supports which can result
in cleaner backgrounds and help for samples such as small
proteins or thin two-dimensional (2D) crystals.
4. UF Solution: The volumes used in making UF can be adjusted
accordingly along with the amount of 5 M NaOH.
5. Crystals of the stain can be observed on the microscope when
precipitation of the stain occurs. This could hinder screening
for crystalline samples and can affect the quality of the staining
and usable areas for data collection.
6. The protocol can be optimized in many ways depending on the
results seen on the microscope, for example:
Concentration of sample on the grid: Adjust sample volume,
adsorption time, glow-discharge time, add chemicals to change
the surface properties of the grid, etc.
Flattening: Adjust thickness of the staining, skip the water
wash in order to stain faster, skip the vacuum aspiration.
7. While I recommend water as a default step for washing, some
samples may need either a buffer wash (if water is not favored)
or even skipping the wash step completely.
8. Grids with intact or mostly intact carbon will be able to contain
all the solutions in the staining procedure to the carbon side of
the grid. If a solution is observed on the opposite side, most or
all of the carbon support has been broken and has allowed the
solution to penetrate the grid. If the grid can be re-stained, it is
recommended to do so, if not, check the grid on the microscope, there may be enough usable area to answer the questions
required and/or collect data for processing.
9. While electron dose is not a major factor in a negative-stain
experiment as in CryoEM, areas being imaged can burn and
carbon supports can break. For data collection, it is best to
image away from screening and beam-alignment areas.
10. When the concentration of the sample is too high, the particles
can cluster and not stain well, resulting in a lack in definition.
Low concentrations can result in large grid areas of only background carbon. A well-dispersed sample will fill each micrograph with many particles but still be individually well defined
with space between each particle (Figs. 1 and 3). For cases
involving fibers or crystals, the translation of concentration to
a grid can be difficult to establish. Long fibers for example can
bunch up in certain areas of the grid and may need to be
broken up for dispersal and crystals can contain different
amounts of repeat units, sometimes numbering in the
thousands.
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Shane Gonen
basis—detailed protocols are available [15]. Coating grids
allows the user to create thin carbon supports which can result
in cleaner backgrounds and help for samples such as small
proteins or thin two-dimensional (2D) crystals.
4. UF Solution: The volumes used in making UF can be adjusted
accordingly along with the amount of 5 M NaOH.
5. Crystals of the stain can be observed on the microscope when
precipitation of the stain occurs. This could hinder screening
for crystalline samples and can affect the quality of the staining
and usable areas for data collection.
6. The protocol can be optimized in many ways depending on the
results seen on the microscope, for example:
Concentration of sample on the grid: Adjust sample volume,
adsorption time, glow-discharge time, add chemicals to change
the surface properties of the grid, etc.
Flattening: Adjust thickness of the staining, skip the water
wash in order to stain faster, skip the vacuum aspiration.
7. While I recommend water as a default step for washing, some
samples may need either a buffer wash (if water is not favored)
or even skipping the wash step completely.
8. Grids with intact or mostly intact carbon will be able to contain
all the solutions in the staining procedure to the carbon side of
the grid. If a solution is observed on the opposite side, most or
all of the carbon support has been broken and has allowed the
solution to penetrate the grid. If the grid can be re-stained, it is
recommended to do so, if not, check the grid on the microscope, there may be enough usable area to answer the questions
required and/or collect data for processing.
9. While electron dose is not a major factor in a negative-stain
experiment as in CryoEM, areas being imaged can burn and
carbon supports can break. For data collection, it is best to
image away from screening and beam-alignment areas.
10. When the concentration of the sample is too high, the particles
can cluster and not stain well, resulting in a lack in definition.
Low concentrations can result in large grid areas of only background carbon. A well-dispersed sample will fill each micrograph with many particles but still be individually well defined
with space between each particle (Figs. 1 and 3). For cases
involving fibers or crystals, the translation of concentration to
a grid can be difficult to establish. Long fibers for example can
bunch up in certain areas of the grid and may need to be
broken up for dispersal and crystals can contain different
amounts of repeat units, sometimes numbering in the
thousands.
122
Shane Gonen