21. The smooth (“shiny”) side of the grid is facing up and the
straight leg of the anticapillary forceps is facing down.
22. Only continuous, unbroken carbon film is used for the
next step.
23. The smooth side of the grid is still facing up for this step.
24. Success using this grid preparation method depends greatly on
using 600-mesh (or finer) EM grids. Standard 400-mesh grids
do not provide enough support for the carbon film during
capillary pressure adhesion, leading to an unacceptable amount
of carbon film breakage. Grids should be pre-cleaned by rinsing
in chloroform and dried completely. As this method causes
significant stress to the carbon film, it is also imperative that
the film be solid “spark-free” carbon film, without any defects
that could eventually result in breakage. It can be advantageous
to inspect the carbon film carefully as it is initially floated onto
buffer, checking the reflection of a light off the carbon/buffer
surface to check for cracks, embedded particles, etc.
25. To utilize this method for preparation of negatively stained
samples, buffer should contain negative stain, e.g., 1–2% uranyl
acetate or uranyl formate.
26. It is important to keep the grid parallel to the membrane
surface during placement, and for the EM grid to make full
contact with the membrane surface, ensuring a good seal for
step 4 below.
27. Millipore DAWP membrane filters absorb aqueous buffer at a
much slower rate than Whatman paper, meaning that somewhat long blotting times are needed. From the time of initial
contact with the membrane, it can take 1–5 s for the carbon
film to become completely flat on the surface of the grid. In
addition, for the capillary pressure to sufficiently bring the EM
grid surface and the carbon film into close contact takes a
further 2–3 s. The best results were observed by blotting
until 3 s after the carbon film had completely flattened.
28. The grid should be quickly moved onto the droplet after lifting
from the membrane to ensure that the surface does not dry
out. In some cases, the carbon film will adhere tightly to the
grid, and it can be helpful to use a second pair of forceps to
gently press the grid down onto parafilm, forcing buffer
through the grid mesh.
29. This may be helpful when the stacked crystals are many layers
thick, with the increased risk of damage that accompanies
increased manipulation.
30. For negatively stained samples, air dry only (omit plunging).
2D Electron Crystallography of Membrane Proteins
243
straight leg of the anticapillary forceps is facing down.
22. Only continuous, unbroken carbon film is used for the
next step.
23. The smooth side of the grid is still facing up for this step.
24. Success using this grid preparation method depends greatly on
using 600-mesh (or finer) EM grids. Standard 400-mesh grids
do not provide enough support for the carbon film during
capillary pressure adhesion, leading to an unacceptable amount
of carbon film breakage. Grids should be pre-cleaned by rinsing
in chloroform and dried completely. As this method causes
significant stress to the carbon film, it is also imperative that
the film be solid “spark-free” carbon film, without any defects
that could eventually result in breakage. It can be advantageous
to inspect the carbon film carefully as it is initially floated onto
buffer, checking the reflection of a light off the carbon/buffer
surface to check for cracks, embedded particles, etc.
25. To utilize this method for preparation of negatively stained
samples, buffer should contain negative stain, e.g., 1–2% uranyl
acetate or uranyl formate.
26. It is important to keep the grid parallel to the membrane
surface during placement, and for the EM grid to make full
contact with the membrane surface, ensuring a good seal for
step 4 below.
27. Millipore DAWP membrane filters absorb aqueous buffer at a
much slower rate than Whatman paper, meaning that somewhat long blotting times are needed. From the time of initial
contact with the membrane, it can take 1–5 s for the carbon
film to become completely flat on the surface of the grid. In
addition, for the capillary pressure to sufficiently bring the EM
grid surface and the carbon film into close contact takes a
further 2–3 s. The best results were observed by blotting
until 3 s after the carbon film had completely flattened.
28. The grid should be quickly moved onto the droplet after lifting
from the membrane to ensure that the surface does not dry
out. In some cases, the carbon film will adhere tightly to the
grid, and it can be helpful to use a second pair of forceps to
gently press the grid down onto parafilm, forcing buffer
through the grid mesh.
29. This may be helpful when the stacked crystals are many layers
thick, with the increased risk of damage that accompanies
increased manipulation.
30. For negatively stained samples, air dry only (omit plunging).
2D Electron Crystallography of Membrane Proteins
243