2. The small reservoir that provides a large water surface can be
used to hold the embedding solution or mother buffer. The
larger surface area will help one to pick up the carbon film.
3. Molybdenum grids are usually used in electron crystallography
to reduce the cryo-crinkling effect [50], which is a phenomenon that the support carbon film wrinkles when the grid is
cooled down to the liquid nitrogen temperatures. The wrinkling is due to different rates of thermal expansion between the
grid metal and the carbon film. The thermal expansion coefficient of the molybdenum is two to three times less than that of
copper, and molybdenum is thus a better material than copper
for reducing the wrinkling of carbon film [50].
4. In this protocol, we will use an Oxford CT3500 side-entry or a
Gatan 626 cryo-specimen holder as an example. The actual
procedure of the specimen transfer depends on the stage design
of the TEM used.
5. The quality of the carbon film is critical to the success of grid
specimen preparation. When evaporating the carbon, make
sure to slowly and continuously deposit the carbon on top of
the mica sheet.
6. Sparking usually occurs when the current increases too fast and
leads to a strong discharging from the carbon source. If the
sparks are observed, the carbon film is possibly broken and will
be needed an inspection under a light microscope.
7. Before applying the 2D crystal sample onto the grid, one may
need to pipet a few times in the crystal suspension because the
membrane crystals are easy to cluster while sitting in the refrigerator. Gently pipetting ensures an appropriate amount of the
crystal sample when preparing the EM grid specimen.
8. If one reuses the molybdenum grids, the grids are needed to be
cleaned before use. The used grids can be cleaned in chloroform, ethanol, and deionized water with sonication. Before
working on specimen preparation, one or two rounds of plasma
cleaning on the grid is recommended.
9. Sugar may not be evenly distributed on the grid and may form a
small size of the crystals that are distinguishable especially in
the diffraction pattern. It shows a diffraction pattern of a
hexagonal lattice overlaid on the diffraction pattern of the 2D
protein crystal. Mixing the crystal sample thoroughly with the
embedding solution may help to alleviate this problem. On the
other hand, the intensities from the sugar crystalline are strong
and can be visible in the diffraction pattern (Fig. 7). These
reflections can be masked out before the background subtraction and have a minimal effect on the integration of the diffraction intensities of the 2D crystal.
262
Ka-Yi Chan et al.
used to hold the embedding solution or mother buffer. The
larger surface area will help one to pick up the carbon film.
3. Molybdenum grids are usually used in electron crystallography
to reduce the cryo-crinkling effect [50], which is a phenomenon that the support carbon film wrinkles when the grid is
cooled down to the liquid nitrogen temperatures. The wrinkling is due to different rates of thermal expansion between the
grid metal and the carbon film. The thermal expansion coefficient of the molybdenum is two to three times less than that of
copper, and molybdenum is thus a better material than copper
for reducing the wrinkling of carbon film [50].
4. In this protocol, we will use an Oxford CT3500 side-entry or a
Gatan 626 cryo-specimen holder as an example. The actual
procedure of the specimen transfer depends on the stage design
of the TEM used.
5. The quality of the carbon film is critical to the success of grid
specimen preparation. When evaporating the carbon, make
sure to slowly and continuously deposit the carbon on top of
the mica sheet.
6. Sparking usually occurs when the current increases too fast and
leads to a strong discharging from the carbon source. If the
sparks are observed, the carbon film is possibly broken and will
be needed an inspection under a light microscope.
7. Before applying the 2D crystal sample onto the grid, one may
need to pipet a few times in the crystal suspension because the
membrane crystals are easy to cluster while sitting in the refrigerator. Gently pipetting ensures an appropriate amount of the
crystal sample when preparing the EM grid specimen.
8. If one reuses the molybdenum grids, the grids are needed to be
cleaned before use. The used grids can be cleaned in chloroform, ethanol, and deionized water with sonication. Before
working on specimen preparation, one or two rounds of plasma
cleaning on the grid is recommended.
9. Sugar may not be evenly distributed on the grid and may form a
small size of the crystals that are distinguishable especially in
the diffraction pattern. It shows a diffraction pattern of a
hexagonal lattice overlaid on the diffraction pattern of the 2D
protein crystal. Mixing the crystal sample thoroughly with the
embedding solution may help to alleviate this problem. On the
other hand, the intensities from the sugar crystalline are strong
and can be visible in the diffraction pattern (Fig. 7). These
reflections can be masked out before the background subtraction and have a minimal effect on the integration of the diffraction intensities of the 2D crystal.
262
Ka-Yi Chan et al.