added 4 h after APol addition or the following day by mixing on an end-to-end rotator at
4
C.
The time at which to add Bio-Beads is a protocol optimization point that needs to be
empirically determined. For TRPV1, Bio-Beads are added 4 h after APols. For TRPA1,
Bio-Beads are added the following morning because earlier Bio-Bead addition results in
detectable loss of protein features as assessed by negative stain electron microscopy. If it is
necessary to remove the MBP affinity tag, add an empirically determined amount of TEV
protease (~1:25 w/w ratio to the MP) along with the APol and mix overnight on an end-toend rotator.
12. Remove Bio-Beads over a disposable Poly-Prep column.
13. Clear eluent by centrifugation (14,000 rpm, 5 min, 4
C) before further purification on a
Superose 6 or Superdex 200 column pre-equilibrated in gel filtration buffer.
For TEV-digested TRPV1 (300 kDa tetramer) and MBP-tagged TRPA1 (690 kDa tetramer), biochemically well-behaved, homogeneous protein elutes around 11 mL on a
Superdex 200 and around 13 mL on a Superose 6 column, respectively. Both columns
have a 25 mL bed volume. When PMAL-C8 is used as the APol, excess PMAL-C8 elutes as a
separate peak around 17 mL on a Superose 6 column as detected with the in-line A 280
detector. A corresponding peak is not seen with excess A8-35, which does not absorb at this
wavelength (see Chaps. 4 and 8).
14. Combine protein-containing fractions and concentrate to 0.5–3 g‧L
À1 in a 100 kDa cutoff
Amicon filter by centrifugation (3000 rpm, 7 min, 4
C).
15. Use sample to prepare grids for cryo-EM analysis. Many aspects of freezing cryo-grids must
be empirically determined including the recourse to additives (e.g. glycerol, agonists,
antagonists), the optimal protein concentration applied to the grids (could be anywhere
from 0.5 to 3 g‧L
À1 ), the volume of sample applied to the grids (e.g. 2.5 μL), blotting time,
type of cryo-grids used (e.g. 200-mesh or 400-mesh Quantifoil grids), glow discharge time,
and relative humidity in the blotting chamber.
Wishing you all the very best of luck on your membrane protein adventures,
Candice E. Paulsen, Department of Physiology, University of California, San Francisco. Protocol based on Cao et al. (2013a), Liao et al. (2013), and Paulsen et al. (2015) (Studies 12.9 and 12.21),
with added comments.
References
Abeyrathne, P.D., Grigorieff, N. (2017) Expression, purification, and contaminant detection for structural studies of
Ralstonia metallidurance ClC protein rm1. PLoS One 12:e0180163.
Agard, D.A., Cheng, Y., Glaeser, R.M., Subramaniam, S. (2014) Single-particle cryo-electron microscopy (cryo-EM):
progress, challenges, and perspectives for future improvement. Adv. Imaging Electron Phys. 185:113–137.
Agosto, M.A., Zhang, Z., He, F., Anastassov, I.A., Wright, S.J., McGehee, J., Wensel, T.G. (2014) Oligomeric state of
purified TRPM1, a protein essential for dim light vision. J. Biol. Chem. 289:27019–27033.
Allegretti, M., Mills, D.J., McMullan, G., Kühlbrandt, W., Vonck, J. (2014) Atomic model of the F 420 -reducing [NiFe]
hydrogenase by electron cryo-microscopy using a direct electron detector. eLife 3:e01963.
References
583
4
C.
The time at which to add Bio-Beads is a protocol optimization point that needs to be
empirically determined. For TRPV1, Bio-Beads are added 4 h after APols. For TRPA1,
Bio-Beads are added the following morning because earlier Bio-Bead addition results in
detectable loss of protein features as assessed by negative stain electron microscopy. If it is
necessary to remove the MBP affinity tag, add an empirically determined amount of TEV
protease (~1:25 w/w ratio to the MP) along with the APol and mix overnight on an end-toend rotator.
12. Remove Bio-Beads over a disposable Poly-Prep column.
13. Clear eluent by centrifugation (14,000 rpm, 5 min, 4
C) before further purification on a
Superose 6 or Superdex 200 column pre-equilibrated in gel filtration buffer.
For TEV-digested TRPV1 (300 kDa tetramer) and MBP-tagged TRPA1 (690 kDa tetramer), biochemically well-behaved, homogeneous protein elutes around 11 mL on a
Superdex 200 and around 13 mL on a Superose 6 column, respectively. Both columns
have a 25 mL bed volume. When PMAL-C8 is used as the APol, excess PMAL-C8 elutes as a
separate peak around 17 mL on a Superose 6 column as detected with the in-line A 280
detector. A corresponding peak is not seen with excess A8-35, which does not absorb at this
wavelength (see Chaps. 4 and 8).
14. Combine protein-containing fractions and concentrate to 0.5–3 g‧L
À1 in a 100 kDa cutoff
Amicon filter by centrifugation (3000 rpm, 7 min, 4
C).
15. Use sample to prepare grids for cryo-EM analysis. Many aspects of freezing cryo-grids must
be empirically determined including the recourse to additives (e.g. glycerol, agonists,
antagonists), the optimal protein concentration applied to the grids (could be anywhere
from 0.5 to 3 g‧L
À1 ), the volume of sample applied to the grids (e.g. 2.5 μL), blotting time,
type of cryo-grids used (e.g. 200-mesh or 400-mesh Quantifoil grids), glow discharge time,
and relative humidity in the blotting chamber.
Wishing you all the very best of luck on your membrane protein adventures,
Candice E. Paulsen, Department of Physiology, University of California, San Francisco. Protocol based on Cao et al. (2013a), Liao et al. (2013), and Paulsen et al. (2015) (Studies 12.9 and 12.21),
with added comments.
References
Abeyrathne, P.D., Grigorieff, N. (2017) Expression, purification, and contaminant detection for structural studies of
Ralstonia metallidurance ClC protein rm1. PLoS One 12:e0180163.
Agard, D.A., Cheng, Y., Glaeser, R.M., Subramaniam, S. (2014) Single-particle cryo-electron microscopy (cryo-EM):
progress, challenges, and perspectives for future improvement. Adv. Imaging Electron Phys. 185:113–137.
Agosto, M.A., Zhang, Z., He, F., Anastassov, I.A., Wright, S.J., McGehee, J., Wensel, T.G. (2014) Oligomeric state of
purified TRPM1, a protein essential for dim light vision. J. Biol. Chem. 289:27019–27033.
Allegretti, M., Mills, D.J., McMullan, G., Kühlbrandt, W., Vonck, J. (2014) Atomic model of the F 420 -reducing [NiFe]
hydrogenase by electron cryo-microscopy using a direct electron detector. eLife 3:e01963.
References
583