5. Vortex the sample for 10–20 s.
6. Incubate the dialysate for 10 min on ice. During the incubation, rinse the dialysis tubing with ultrapure water and divide
the dialysis buffer into four beakers (400 mL volume).
7. Vortex the dialysate for 10–20 s.
8. Attach a dialysis clip to one end of the dialysis tubing, leaving at
least several mm to 1 cm on the bottom end of the clip.
9. Remove excess water from the inside of the tubing by holding
the clip and rapidly flicking the long end of the tubing.
10. Hold the clip between the thumb and ring finger of one hand
and stabilize the long, open end of the tubing between the
index and middle finger in order to hold the tubing upright.
11. Pipette the dialysate in the open end of the tubing, ensuring
that the pipette tip is placed as closely as possible to the bottom
clip before pipetting the dialysate (see Note 6).
12. With a second dialysis clip placed at an angle of 90
, clip the
open end of the tubing closed, while keeping a space of
~6–7 cm between the clips (see Note 7).
13. Place the assembled tubing with one dialysate condition per
beaker in a beaker covered with aluminum foil (see Note 8).
14. Dialyze the sample for 2–14 days in an incubator at a temperature just above the phase transition temperature of the lipid (see
Note 9).
15. Remove the sample from the incubator and cut the dialysis
tubing below the top clip.
16. Remove the dialysate with a micropipette and place it in an
Eppendorf tube. Immediately proceed to Subheading 3.2
unless the dialysate contains air bubbles (see Note 10).
3.2 Negative Stain
TEM Screening for 2D
Crystallization
Conditions
Screening for 2D crystallization conditions by negative stain TEM
will provide important feedback for the protein purification as well.
Negative stain preparation of the purified protein before dialysis can
provide critical information on, e.g., aggregation or sample
heterogeneity.
3.2.1 Negative Staining
1. A carbon-coated 400-mesh grid is picked up with a pair of
anticapillary forceps with the carbon-side facing up (see
Note 11).
2. A volume of 2 μL of dialysate is pipetted onto the carbon-side
of the grid, incubated for 60 s, and blotted with the torn side of
the filter paper (see Note 12).
3. A same volume of 2 μL 1% uranyl acetate is immediately
pipetted on the grid, the stain is incubated for 30 s on the
232
Matthew C. Johnson et al.
6. Incubate the dialysate for 10 min on ice. During the incubation, rinse the dialysis tubing with ultrapure water and divide
the dialysis buffer into four beakers (400 mL volume).
7. Vortex the dialysate for 10–20 s.
8. Attach a dialysis clip to one end of the dialysis tubing, leaving at
least several mm to 1 cm on the bottom end of the clip.
9. Remove excess water from the inside of the tubing by holding
the clip and rapidly flicking the long end of the tubing.
10. Hold the clip between the thumb and ring finger of one hand
and stabilize the long, open end of the tubing between the
index and middle finger in order to hold the tubing upright.
11. Pipette the dialysate in the open end of the tubing, ensuring
that the pipette tip is placed as closely as possible to the bottom
clip before pipetting the dialysate (see Note 6).
12. With a second dialysis clip placed at an angle of 90
, clip the
open end of the tubing closed, while keeping a space of
~6–7 cm between the clips (see Note 7).
13. Place the assembled tubing with one dialysate condition per
beaker in a beaker covered with aluminum foil (see Note 8).
14. Dialyze the sample for 2–14 days in an incubator at a temperature just above the phase transition temperature of the lipid (see
Note 9).
15. Remove the sample from the incubator and cut the dialysis
tubing below the top clip.
16. Remove the dialysate with a micropipette and place it in an
Eppendorf tube. Immediately proceed to Subheading 3.2
unless the dialysate contains air bubbles (see Note 10).
3.2 Negative Stain
TEM Screening for 2D
Crystallization
Conditions
Screening for 2D crystallization conditions by negative stain TEM
will provide important feedback for the protein purification as well.
Negative stain preparation of the purified protein before dialysis can
provide critical information on, e.g., aggregation or sample
heterogeneity.
3.2.1 Negative Staining
1. A carbon-coated 400-mesh grid is picked up with a pair of
anticapillary forceps with the carbon-side facing up (see
Note 11).
2. A volume of 2 μL of dialysate is pipetted onto the carbon-side
of the grid, incubated for 60 s, and blotted with the torn side of
the filter paper (see Note 12).
3. A same volume of 2 μL 1% uranyl acetate is immediately
pipetted on the grid, the stain is incubated for 30 s on the
232
Matthew C. Johnson et al.