7. Turn the grid upside town (so that the carbon and sample side
are facing down) and quickly touch the grid on the surface of
the first water drop. Quickly remove the excess as per the last
step and continue for all water drops. For samples with detergent, stop after the detergent has been removed by this process—you can visually see when this occurs as the drops
containing detergent will be more opaque than clean Milli-Q.
8. Repeat step 7 with the first stain drop.
9. For the second stain drop, touch the grid on the surface of the
stain but keep the grid touching the stain. Carefully lift the grid
just enough as to not break the surface tension between the
stain and grid and gently move the grid side to side and up and
down. Do this step for 15–20 s and blot the excess as
previously.
10. Air dry the grid and store in a grid holder. For best results,
remove the remaining stain using a vacuum setup with a fine tip
on the edge of the grid for a few seconds.
11. Once stained, grids can be stored for years at room temperature. Refer to standard procedures for imaging (see Notes 9
and 10).
4 Notes
1. Transitioning from negative-stain to CryoEM: This is largely
sample dependent. First step is to access everything you know
about the protein biochemically, for example, is it a multimeric
protein and are all the components present? Is the protein
homogeneous? Second is to estimate conditions for CryoEM
from the negative-stain results. For example, concentration
estimation: CryoEM requires more sample. For single proteins
that are found to be highly concentrated, but still well dispersed, on a negative-stain grid, a good estimate to start
CryoEM with is ~5–10Â the concentration. This can vary
between crystals, fibers, single-proteins, etc.
2. 200 vs. 300/400 copper-mesh grids: There are different
aspects to consider when choosing between 200 mesh copper
grids and 300 or 400, for example, the integrity of the carbon
support. Thinner carbon can easily break on a 200-mesh grid
when compared with 400, leading to less usable areas. Thick
carbon will hold well on 200 mesh. Most single-particle experiments will work well with the smaller areas in a 400-mesh grid
while larger samples such as fibers and crystals may benefit from
the larger areas afforded by a 200-mesh grid.
3. Carbon-coated grids. Copper-mesh grids can be purchased
with or without a pre-coated carbon support. For some
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
121
are facing down) and quickly touch the grid on the surface of
the first water drop. Quickly remove the excess as per the last
step and continue for all water drops. For samples with detergent, stop after the detergent has been removed by this process—you can visually see when this occurs as the drops
containing detergent will be more opaque than clean Milli-Q.
8. Repeat step 7 with the first stain drop.
9. For the second stain drop, touch the grid on the surface of the
stain but keep the grid touching the stain. Carefully lift the grid
just enough as to not break the surface tension between the
stain and grid and gently move the grid side to side and up and
down. Do this step for 15–20 s and blot the excess as
previously.
10. Air dry the grid and store in a grid holder. For best results,
remove the remaining stain using a vacuum setup with a fine tip
on the edge of the grid for a few seconds.
11. Once stained, grids can be stored for years at room temperature. Refer to standard procedures for imaging (see Notes 9
and 10).
4 Notes
1. Transitioning from negative-stain to CryoEM: This is largely
sample dependent. First step is to access everything you know
about the protein biochemically, for example, is it a multimeric
protein and are all the components present? Is the protein
homogeneous? Second is to estimate conditions for CryoEM
from the negative-stain results. For example, concentration
estimation: CryoEM requires more sample. For single proteins
that are found to be highly concentrated, but still well dispersed, on a negative-stain grid, a good estimate to start
CryoEM with is ~5–10Â the concentration. This can vary
between crystals, fibers, single-proteins, etc.
2. 200 vs. 300/400 copper-mesh grids: There are different
aspects to consider when choosing between 200 mesh copper
grids and 300 or 400, for example, the integrity of the carbon
support. Thinner carbon can easily break on a 200-mesh grid
when compared with 400, leading to less usable areas. Thick
carbon will hold well on 200 mesh. Most single-particle experiments will work well with the smaller areas in a 400-mesh grid
while larger samples such as fibers and crystals may benefit from
the larger areas afforded by a 200-mesh grid.
3. Carbon-coated grids. Copper-mesh grids can be purchased
with or without a pre-coated carbon support. For some
Progress Towards CryoEM: Negative-Stain Procedures for Biological Samples
121