A lipid mix consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (6:4 w/w, Anatrace) in chloroform was dried under nitrogen gas and rehydrated in buffer containing 20 mM HEPES-NaOH, 150 mM NaCl at pH 7.4. Rehydrated liposomes were extruded through a 100 nm polycarbonate membrane (Whatman) to produce a monodisperse solution of unilamellar liposomes, whose local membrane curvature is negligible on the length scale of the complete pore. Complement proteins C5b6, C7, C8, and C9 (Complement Technology) were sequentially incubated with liposomes at a molar ratio of 1:1:1:18. A 5 min incubation step was allowed between each component addition followed by incubation for 1 h at 37 °C to optimize for assembly completion before transferring to 4 °C overnight. Assembled MAC complexes were solubilized in 1.5 % Cymal-5 (Anatrace) in the presence of DOPC (1 mg/ml) and glycerol (10 %) for 1 hour at room temperature. Solubilized complexes were purified using density centrifugation in a sucrose solution (5–20%) containing 0.004 % Cymal-7 NG (Anatrace). Samples were spun for 4 hours at 45,000 rpm using an SW60Ti rotor. Fractions were screened using negative stain EM and those containing complete MAC pores were pooled, concentrated and sucrose removed using a ZebaSpin desalting column (Thermofisher Scientific).
Cymal 5
Manufactured by Anatrace
Sourced in Japan
Cymal-5 is a non-ionic detergent used for the solubilization and purification of membrane proteins. It is a mild detergent with a critical micelle concentration (CMC) of approximately 0.6 mM.
Automatically generated - may contain errors
Lab products found in correlation
100 nm polycarbonate membrane, by Cytiva (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Tween 20, by Fujifilm (1 mentions)
Triton x 100, by Fujifilm (1 mentions)
N dodecyl β d maltoside ddm, by Dojindo Laboratories (1 mentions)
N dodecylphosphocholine dpc, by Anatrace (1 mentions)
Chaps, by Dojindo Laboratories (1 mentions)
Autoflex 3 smartbeam, by Bruker (1 mentions)
3 protocols using cymal 5
1
Reconstitution of Membrane Attack Complex
2
Solubilization and Purification of His-MotA Protein
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The membrane fraction containing the His-MotAAa protein was solubilized in 1% (w/v) of CHAPS (Dojindo, Kamimashiki, Japan), Cymal-5 (Anatrace, Maumee, OH, USA), n-dodecyl β-d -maltoside (DDM) (Dojindo), n-decyl β-d -maltoside (DM) (Dojindo), n-dodecylphosphocholine (DPC) (Anatrace), n-octyl β-d -glucoside (OG) (Dojindo), sucrose monocaprate (SMC) (Dojindo), Triton-X100 (Wako) or Tween-20 (Wako) for about 30 min on ice. The detergent solubilized membrane sample was ultra-centrifuged at 100 000 × g for 30 min, and separated into pellet (insoluble fraction) and supernatant (soluble fraction). The proteins were resolved by SDS-PAGE and subsequently detected by CBB staining and immunoblot analysis, using the anti-His antibody (MBL, Nagoya, Japan).
3
Competitive Inhibition of ATP Synthase
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Purified M. phlei c-ring in 0.6% Cymal-5 (Anatrace) was used to assay the competition of DCCD with BDQ. The concentrated sample (15 mg/ml) was diluted to 0.1 mg/ml using 20 mM cacodylate/trimethylamine/NH3 (pH 7.5). BDQ, solubilized in dimethyl sulfoxide, was added to the indicated final concentrations, and samples were incubated for 1 hour at room temperature before adding 25 μM DCCD. Aliquots were removed at several time points (5 to 60 min), directly mixed in a 1:1 ratio with 2′,4′,-dihydroxyacetophenone matrix, and applied onto a ground steel MALDI target in duplicates. Mass spectra were acquired on a MALDI–time-of-flight (TOF)/TOF mass spectrometer (Bruker Autoflex III Smartbeam) and evaluated as previously described (32 (link)). Error bars were calculated from four individual measurements. Graphs and curves were generated using SigmaPlot.
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