An alternative and quicker purification protocol that serves as a protocol optimization
point and which works equally well for TRPV1 and TRPA1 is to directly flash-freeze cell
pellets (before resuspension) and store at À80
C before purification. The cell pellets are
thawed in a water bath at room temperature, resuspended in membrane resuspension buffer
without glycerol (25 mL per liter of cells), further resuspended with a Dounce homogenizer,
and directly solubilized by adding the appropriate detergent as in step 8. The subsequent
purification protocol is then continued forward.
5. Clear cell debris by centrifugation (8000Â g, 20 min, 4
C).
6. Transfer supernatant to ultracentrifuge tubes and collect membranes by ultracentrifugation in
a Beckman 45 Ti rotor (200,000Â g, 60 min, 4
C).
7. Discard supernatant and resuspend membrane pellets in membrane resuspension buffer
(25 mL buffer for each liter of cells) on ice. Use a Dounce homogenizer to aid membrane
resuspension.
Membranes can be prepared in batch, flash-frozen in liquid nitrogen, and stored at À80
C
at this stage for future purifications. Of note, we have found that resuspended membranes are
not as stable over time (longer than 1 year) as cell pellets.
8. Thaw resuspended membranes in a room-temperature water bath. Solubilize membranes with
20 mM DDM or 10 mM MNG-3 on an end-to-end rotator for 2 h at 4
C.
The optimal detergent for purification needs to be empirically determined for each MP of
interest. The fluorescence size exclusion chromatography (FSEC) procedure developed by
the laboratory of Eric Gouaux represents an excellent screening strategy (Kawate and
Gouaux 2006). Detergent extraction time and extraction temperature (4
C vs. room temperature) are additional points of optimization. Often times, complete extraction can be accomplished in 1–1.5 h at 4
C.
9. Remove detergent-insoluble material by ultracentrifugation in a Beckman 45 Ti rotor
(30,000Â g, 30 min, 4
C) and mix the supernatant with amylose resin (1 mL of 50% slurry
per liter of cells) on an end-to-end rotator for 2 h at 4
C.
The amylose resin is supplied in an ethanol solution. A working stock of resin is prepared
as a 50% slurry in water by washing the resin 3Â with water. Pellet resin at 1000 rpm
between washes. Store working resin stock at 4
C.
10. Collect resin in a disposable Poly-Prep column at room temperature. Wash resin with ten
column volumes of wash buffer and elute bound protein with elution buffer (200 μL
fractions for each 1 mL of 50% resin slurry). Measure A 280 and pool fractions containing
protein.
Measuring A 280 can be achieved in a number of ways. We use a NanoDrop.
11. Mix pooled protein with APols (Anatrace, A8-35 or PMAL-C8, 100 g‧L
À1 or 50 g‧L
À1
stock solutions freshly prepared in water, respectively) at a 1:3 (w/w) dilution (MP:APol) on
an end-to-end rotator overnight at 4
C. Remove detergent with Bio-Beads SM-2 (Bio-Rad)
582
12 The Use of Amphipols for Electron Microscopy
point and which works equally well for TRPV1 and TRPA1 is to directly flash-freeze cell
pellets (before resuspension) and store at À80
C before purification. The cell pellets are
thawed in a water bath at room temperature, resuspended in membrane resuspension buffer
without glycerol (25 mL per liter of cells), further resuspended with a Dounce homogenizer,
and directly solubilized by adding the appropriate detergent as in step 8. The subsequent
purification protocol is then continued forward.
5. Clear cell debris by centrifugation (8000Â g, 20 min, 4
C).
6. Transfer supernatant to ultracentrifuge tubes and collect membranes by ultracentrifugation in
a Beckman 45 Ti rotor (200,000Â g, 60 min, 4
C).
7. Discard supernatant and resuspend membrane pellets in membrane resuspension buffer
(25 mL buffer for each liter of cells) on ice. Use a Dounce homogenizer to aid membrane
resuspension.
Membranes can be prepared in batch, flash-frozen in liquid nitrogen, and stored at À80
C
at this stage for future purifications. Of note, we have found that resuspended membranes are
not as stable over time (longer than 1 year) as cell pellets.
8. Thaw resuspended membranes in a room-temperature water bath. Solubilize membranes with
20 mM DDM or 10 mM MNG-3 on an end-to-end rotator for 2 h at 4
C.
The optimal detergent for purification needs to be empirically determined for each MP of
interest. The fluorescence size exclusion chromatography (FSEC) procedure developed by
the laboratory of Eric Gouaux represents an excellent screening strategy (Kawate and
Gouaux 2006). Detergent extraction time and extraction temperature (4
C vs. room temperature) are additional points of optimization. Often times, complete extraction can be accomplished in 1–1.5 h at 4
C.
9. Remove detergent-insoluble material by ultracentrifugation in a Beckman 45 Ti rotor
(30,000Â g, 30 min, 4
C) and mix the supernatant with amylose resin (1 mL of 50% slurry
per liter of cells) on an end-to-end rotator for 2 h at 4
C.
The amylose resin is supplied in an ethanol solution. A working stock of resin is prepared
as a 50% slurry in water by washing the resin 3Â with water. Pellet resin at 1000 rpm
between washes. Store working resin stock at 4
C.
10. Collect resin in a disposable Poly-Prep column at room temperature. Wash resin with ten
column volumes of wash buffer and elute bound protein with elution buffer (200 μL
fractions for each 1 mL of 50% resin slurry). Measure A 280 and pool fractions containing
protein.
Measuring A 280 can be achieved in a number of ways. We use a NanoDrop.
11. Mix pooled protein with APols (Anatrace, A8-35 or PMAL-C8, 100 g‧L
À1 or 50 g‧L
À1
stock solutions freshly prepared in water, respectively) at a 1:3 (w/w) dilution (MP:APol) on
an end-to-end rotator overnight at 4
C. Remove detergent with Bio-Beads SM-2 (Bio-Rad)
582
12 The Use of Amphipols for Electron Microscopy