We prepared micelles by solubilizing a dried lipid film made from DOPC/DOPS (60/40 mol/mol) at 25 °C in 100 mM NaCl, 20 mM Hepes pH = 7.5, 20 mM CHAPS (3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate hydrate, Sigma-Aldrich) at a total lipid concentration of 12 mM. The following protocol is adapted from25 (link). In brief, micelles were homogenized by bath sonication and stirring at 25 °C for 1 h before addition of 4 µM Snf7, 2 µM Vps24 and 2 µM Vps2, making sure that the detergent concentration was above its critical micellar concentration after addition of all proteins. The sample was then gradually diluted four-fold over 30 min under agitation at 25 °C and further incubated for 5 h. For cryo-EM, 4 µL of the sample were deposited on glow-discharged Quantifoil R1.2/1.3 300 mesh copper grids and plunge frozen in liquid ethane after a two-sided blot using a FEI Vitrobot.
3 3 cholamidopropyl dimethyl ammonio 1 propanesulfonate hydrate
Manufactured by Merck Group
Sourced in United States
3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate hydrate is a zwitterionic detergent used in biological research and laboratory applications. It is commonly employed as a mild, non-denaturing solubilizing agent for the extraction and purification of membrane proteins.
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Lab products found in correlation
Chaps, by Merck Group (2 mentions)
Vitrobot, by Thermo Fisher Scientific (1 mentions)
Tecnai g2 sphera lab6, by Thermo Fisher Scientific (1 mentions)
Porcine trypsin, by Promega (1 mentions)
Caffeic acid, by Merck Group (1 mentions)
Ferulic acid, by Merck Group (1 mentions)
Vanillic acid, by Merck Group (1 mentions)
P coumaric acid, by Merck Group (1 mentions)
Syringic acid, by Merck Group (1 mentions)
Pharmalyte, by Cytiva (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Acetonitrile, by Merck Group (1 mentions)
Trifluoroacetic acid, by Merck Group (1 mentions)
Acrylamide, by Merck Group (1 mentions)
Trolox, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Thiourea, by Merck Group (1 mentions)
4 hydroxybenzoic acid, by Merck Group (1 mentions)
6 protocols using 3 3 cholamidopropyl dimethyl ammonio 1 propanesulfonate hydrate
1
Reconstitution of ESCRT Complex
2
Reconstitution of ESCRT-III Helical Tubes
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We prepared micelles by solubilizing a dried lipid film made from DOPC/DOPS (60/40 mol/mol) at 25°C in 100 mM NaCl, 20 mM HEPES pH = 7.5, 20 mM CHAPS (3-[(3-Cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate hydrate, Sigma-Aldrich) at a total lipid concentration of 12 mM. The following protocol is adapted from Szwedziak et al. (2014) (link). In brief, micelles were homogenized by bath sonication and stirring at 25°C for 1 h before addition of 4 μM Snf7, 2 μM Vps24 and 2 μM Vps2, making sure that the detergent concentration was above its critical micellar concentration after addition of all proteins. The sample was then gradually diluted four-fold over 30 min under agitation at 25°C and further incubated for 5 h. For cryo-EM, 4 μL of the sample were deposited on glow-discharged Quantifoil R1.2/1.3 300 mesh copper grids and plunge frozen in liquid ethane after a two-sided blot using a FEI Vitrobot. Vitrified helical tubes were imaged in low-dose mode on a FEI Tecnai G2 Sphera LaB6 at 200 kV using a 4k x 4k FEI Eagle Camera. RELION 2.0.4 was used for 2D classification of manually selected helical segments.
3
Comprehensive Proteomic Analysis Workflow
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Acrylamide, acetonitrile, α-cyano-4-hydroxycinnamic acid, bis-Acrylamide, benzamidine, Bradford solution, p-coumaric acid, caffeic acid, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (CHAPS), 1,4-dithiothreitol (DTT), ferulic acid, fluorescein (FL), γ-aminobutyric acid (GABA), gallic acid, 4-hydroxybenzoic acid, iodoacetamide, phenylisothiocyanate, syringic acid, sodium dodecyl sulfate (SDS), Trolox, trifluoroAcetic acid, thiourea, urea, and vanillic acid were purchased from Sigma Aldrich (St. Louis, MO, USA). Acetic acid, dipotassium phosphate, ethanol, hydrochloric acid, hexane, sodium hydroxide, monopotassium phosphate, methanol, and water were obtained from Junsei Chemical (Tokyo, Japan), and 2,2′-azobis(2-amidinoprpane) dihydrochloride solution (ABAP) was purchased from Wako Chemicals (Richmond, VA, USA). Pharmalyte (pH 3.5–10) and IPG Dry Strips (pH4–10 NL, 24 cm) were purchased from Amersham Biosciences and from Genomine Inc. (Pohang-si, Korea), respectively. Porcine trypsin was obtained from Promega (Madison, WI, USA).
4
Detergent Solubilization of Native KCC2 Protein
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All biochemical preparations and centrifugations were performed at 4°C as previously described (Ivakine et al., 2013 (link); Mahadevan et al., 2014 (link), 2015 (link)). Systematic analysis of detergent solubility, and migration of native-KCC2 from crude membrane fractions were performed according to the workflow described in Figure 1—figure supplement 1 . The following eight detergents (or detergent combinations) were used to solubilize whole brain membranes: C12E9 (1.5%; nonaethylene glycol monododecyl ether, Sigma-Aldrich, St. Louis, MO, #P9641), CHAPS (1.5%; 3-[(3-Cholamidopropyl) dimethylammonio]−1-propanesulfonate hydrate, Sigma-Aldrich #C3023), DDM, (1.5%; DDM, n-dodecyl β-d-maltoside, Sigma-Aldrich #D4641), DOC (1%; sodium deoxycholate, Sigma-Aldrich #D6750), NP40 (1%; nonyl phenoxypolyethoxylethanol, Thermo Fisher Scientific # 28324), Triton-X-100 (1%; 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol, t-Octylphenoxypolyethoxyethanol, Polyethylene glycol tert-octylphenyl ether, Sigma-Aldrich #X-100), Triton-X-100 (1%) +DOC (1%), SDS (0.1%; Sodium dodecyl sulphate, (Sigma-Aldrich #71725) +DOC (1%)+NP40 0.5%).
5
Membrane Protein Extraction and Purification
6
Isolation of Membrane Proteins Containing H-2Kb
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Membrane proteins containing H-2Kb (Kb) were isolated from an EG7.OVA tumor mass. Briefly, 1 g of EG7.OVA tumor mass was dissociated with 10 ml of extraction buffer containing 0.15 M NaCl, 0.05 M sodium phosphate buffer (pH 7.0), and 1X protease inhibitor cocktail (Sigma-Aldrich) using gentlMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). After removing cell debris and lipids, the supernatant was ultra-centrifuged at 80,000 × g for 45 min at 4°C using Ultracentrifuge (Beckman Coulter, Brea, CA, USA). The pellet was homogenized in 0.5 ml of extraction buffer using Tissue Grinder (SHS-30E; SciLab, Seoul, Korea). The homogenate was then mixed with 0.5 ml of solubilization buffer containing 2% n-dodecyl β-D-maltoside and 0.4% 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate hydrate (Sigma-Aldrich) in extraction buffer and solubilized for 90 min at 4°C. The supernatant containing membrane proteins was obtained by centrifugation at 17,000 rpm for 30 min at 4°C. The amount of isolated membrane proteins was determined using a microbicinchoninic acid assay kit (ThermoFisher Scientific, Rockford, IL, USA).
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