9. The dialysis time will depend on the detergent and its concentration. A temperature of 24
C, which is just above the phase
transition temperature is often sufficient for experiments
with DMPC.
10. Air bubbles indicate that a substantial amount of detergent
remains due to an insufficient length of dialysis. The sample
can either be placed in new dialysis tubing (repeat steps 8–16),
or a new dialysate can be prepared to replicate conditions with a
longer dialysis time.
11. Liposomes, proteoliposomes, and 2D crystals will usually
adhere to carbon film without the need for glow-discharge.
12. Volumes of 1.5–5 μL of dialysate may be used. The sample may
be blotted from the top, with the surface of the filter paper
approaching the grid in a parallel manner, especially if the
sample volume is either large and/or concentrated.
13. Several laboratories prefer the use of uranyl formate [41] due
to the close-to-physiological pH and the smaller grain size of
the stain. We prefer uranyl acetate due to the longevity of the
grids after preparation.
14. With longer dialysis timeframes, it is most efficient to screen
the grid as quickly as possible in order to obtain information
for the next dialysis experiment.
15. While 120 kV is standard for screening negatively stained
samples in many cryo-EM laboratories, an accelerating voltage
anywhere in the range of 80–120 kV will work well for
screening.
16. Proteoliposomes can be concentrated at the bottom of an
Eppendorf tube by centrifugation at ~1008 Â g for 3–5 min.
17. Ordered arrays of membrane proteins with large soluble
domains may be identified visually, but FFT assessment will
provide important information such as on crystal stacking,
amount of stacking, mosaicity, first information on plane
group symmetry, and image quality).
18. For assessment of samples before crystallization, it is valuable
to evaluate images with FFTs of both smaller and larger areas
for the best possible signal-to-noise ratio.
19. Ordered arrays of even a few unit cells will provide critical
information for optimization of crystallization conditions.
Thus, a large number of membranes need to be carefully
evaluated.
20. The drops are placed closer to the center of the Parafilm to
avoid them rolling onto the bench when they are manipulated
in the subsequent protocol steps.
242
Matthew C. Johnson et al.
C, which is just above the phase
transition temperature is often sufficient for experiments
with DMPC.
10. Air bubbles indicate that a substantial amount of detergent
remains due to an insufficient length of dialysis. The sample
can either be placed in new dialysis tubing (repeat steps 8–16),
or a new dialysate can be prepared to replicate conditions with a
longer dialysis time.
11. Liposomes, proteoliposomes, and 2D crystals will usually
adhere to carbon film without the need for glow-discharge.
12. Volumes of 1.5–5 μL of dialysate may be used. The sample may
be blotted from the top, with the surface of the filter paper
approaching the grid in a parallel manner, especially if the
sample volume is either large and/or concentrated.
13. Several laboratories prefer the use of uranyl formate [41] due
to the close-to-physiological pH and the smaller grain size of
the stain. We prefer uranyl acetate due to the longevity of the
grids after preparation.
14. With longer dialysis timeframes, it is most efficient to screen
the grid as quickly as possible in order to obtain information
for the next dialysis experiment.
15. While 120 kV is standard for screening negatively stained
samples in many cryo-EM laboratories, an accelerating voltage
anywhere in the range of 80–120 kV will work well for
screening.
16. Proteoliposomes can be concentrated at the bottom of an
Eppendorf tube by centrifugation at ~1008 Â g for 3–5 min.
17. Ordered arrays of membrane proteins with large soluble
domains may be identified visually, but FFT assessment will
provide important information such as on crystal stacking,
amount of stacking, mosaicity, first information on plane
group symmetry, and image quality).
18. For assessment of samples before crystallization, it is valuable
to evaluate images with FFTs of both smaller and larger areas
for the best possible signal-to-noise ratio.
19. Ordered arrays of even a few unit cells will provide critical
information for optimization of crystallization conditions.
Thus, a large number of membranes need to be carefully
evaluated.
20. The drops are placed closer to the center of the Parafilm to
avoid them rolling onto the bench when they are manipulated
in the subsequent protocol steps.
242
Matthew C. Johnson et al.