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Amphipol A8-35

Manufactured by Anatrace
Sourced in United States

Amphipol A8-35 is a non-ionic amphipathic polymer designed for the solubilization and stabilization of membrane proteins. It is composed of a polyacrylate backbone with alkyl side chains, providing both hydrophobic and hydrophilic properties. Amphipol A8-35 can be used to extract and maintain the native structure and function of membrane proteins in aqueous solutions.

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31 protocols using Amphipol A8-35

When BamA protein was required for ELISA or antigen-based sorting, an in vitro biotinylation reaction using BirA enzyme targeting the N-terminal Avi tag was first carried out according to the manufacturers suggestions (Avidity); BamA was then rerun over the Superdex 200 (26/60) in buffer E as described above in order to remove free biotin and other reaction components. When protein was required in amphipol (either non-biotinylated or biotinylated), BamA at 1 mg/mL in buffer E was incubated with a stock solution of 2 mg/mL A8-35 amphipol (Anatrace) at 22 °C for 1 hour and then applied over a Superdex 200 (26/60) column in buffer F (50 mM Tris pH 8, 100 mM NaCl). Protein reconstituted in A8-35 amphipol for immunization was concentrated to 1 mg/mL using a centrifugal device (10 K MWKO; Millipore). When PE-labeled protein was required for antigen-specific sorting, BamA at 1 mg/mL (biotinyated and reconstituted in A8-35 amphipol in buffer F) was mixed 1:1 (vol/vol) with PE-streptavidin (Jackson ImmunoResearch) reconstituted in buffer F; the PE-streptavidin-biotin-BamA-amphipol complex was incubated at 22 °C for at least 15 minutes prior to use.
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When LptDE protein was required for ELISA or antigen-based sorting, an in vitro biotinylation reaction using BirA enzyme targeting the N-terminal Avi tag was first carried out according to the manufacturer's suggestions (Avidity); LptDE was then rerun over the Superdex 200 (26/60) in buffer E as described above in order to remove free biotin and other reaction components. When protein was required in amphipol (either non-biotinylated or biotinylated), LptDE at 1 mg/mL in buffer E was incubated with a stock solution of 2 mg/mL A8-35 amphipol (Anatrace) at 22°C for 1 hr and then applied over a Superdex 200 (26/60) column in buffer F (50 mM Tris pH 8, 100 mM NaCl). Protein reconstituted in A8-35 amphipol for immunization was concentrated to 1 mg/mL using a centrifugal device (10 K MWKO; Millipore). When PE-labeled protein was required for antigen-specific sorting, LptDE at 1 mg/mL (biotinyated and reconstituted in A8-35 amphipol in buffer F) was mixed 1:1 (vol/vol) with PE-streptavidin (Jackson ImmunoResearch) reconstituted in buffer F; the PE-streptavidin-biotin-LptDE-amphipol complex was incubated at 22°C for at least 15 min prior to use.
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A lipid mix containing 1:1 ratio of 1,2-dioleoyl-sn-glycero-3-phosphocoline (DOPC) and cholesteryl hemisuccinate (CHS) was added to the concentrated Env-PGT151 Fab and Env-PGT145 Fab complex such that the final concentration was 0.14 mM. The resulting lipid-Env-Fab mixture (14 μL in total) was incubated on ice for 3 h with 15 Biobeads (Biorad) to partially remove the deoxycholate and the DDM. C-flat holey carbon and gold grids (Electron Microscopy Sciences) were plasma cleaned for 5 s using solarus advanced plasma cleaning system (Gatan) and used for the Env-PGT151 Fab and Env-PGT145 Fab sample. Blotting was performed at 4°C and 100% relative humidity using vitrobot (FEI) as follows. First, 1 μL of amphipol A8-35 (Anatrace) was applied to the grid followed by 3 μL of the Env sample. The grid was blotted for 5 s and plunged into liquid ethane. Grids were stored in liquid nitrogen until use.
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To assemble the HMGCRTM-UBIAD1-BRILN102S-FabBRIL-Nb complex, purified HMGCRTM-UBIAD1-BRILN102S was first incubated with Amphipol A8-35 (Anatrace) for 4 h at 4 °C. The detergent was then removed by overnight incubation with Bio-beads (Bio-Rad). The amphipol-solubilized complex was mixed with FabBRIL and Nb at 1:1.5:2.25 molar ratio for 1 hour at 4 °C. The HMGCRTM-UBIAD1-BRILN102S-FabBRIL-Nb complex was finally purified with a Superose-6 column (GE Healthcare) in buffer A. Peak fractions containing the assembled complex were concentrated to ~10 mg/ml and 2 mM Fluorinated Fos-Choline-8 (Anatrace) was added to the sample before making grids. To assemble the HMGCRTM-UBIAD1-BRILN102S-Fab15B2 complex, purified HMGCRTM-UBIAD1-BRILN102S and Fab15B2 were mixed at 1:1.1 molar ratio and incubated on ice for 1 h, followed by gel-filtration with a Superose-6 column (GE Healthcare) in buffer D. Peak fractions that contained the HMGCRTM-UBIAD1-BRILN102S-Fab15B2 complex were concentrated to ~10 mg/ml. Preparation of HMGCRTM (Δ40–55)-UBIAD1-BRILN102S-Fab15B2 complex sample was following the same procedure.
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Typically, 1 mg each of YaxA and YaxB were combined and incubated at room temperature for 30 min. Cymal-6 was added to 1.5% (w/v) and the mixture incubated at 4 °C for 30 min, after which it was injected onto a Superose 6 column running in buffer E (25 mM HEPES pH 7.0, 150 mM NaCl, 0.05% w/v Cymal-6). Peak fractions were pooled and 3 mg of amphipol A8-35 were (Anatrace) added. The complex was left to incubate for 4 h at 4 °C, after which 20 mg of Bio-Beads (Bio-Rad) were added over night at 4 °C. The next day, amphipol exchanged protein was separated on a Superose 6 column running in detergent free buffer D and used immediately for cryo-EM measurements.
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Purified, concentrated VanSA protein (approx. 150 uM) containing approximately 120 mM LDAO was diluted to 22 uM (1 mg/mL) with 50 mM Tris pH 7.4, 50 mM KCl. An aliquot of a 10% w/v solution of amphipol A8-35 (Anatrace) was added to give a 1:1 mass ratio of protein to amphipol [34 ], and the solution was incubated on ice for 30 minutes. A quantity of washed BioBeads (BioRad) was added equivalent to 20x the estimated mass of LDAO present, and the solution was mixed at 4° for 2 hours. Pilot ultracentrifugation experiments were used to determine that this treatment gave 100% recovery of the VanSA protein in the supernatant. Once this was confirmed, subsequent experiments omitted the ultracentrifugation step. After detergent removal with the BioBeads, the material was injected onto a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with 20 mM Tris-Cl pH 7.4, 150 mM NaCl, and the peak corresponding to active VanSA was collected and used for enzymatic assays. A second, later-eluting peak was also observed, but showed substantially lower activity. The nature of this second species was not investigated further, but we did note that the second peak showed almost no dimer band on SDS-PAGE gels, suggesting it might containing VanSA monomers. Addition of 1% C12E8 to the protein solution prior to amphipol reconstitution decreased the relative size of the second peak.
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Chemicals were from Sigma-Aldrich unless otherwise specified. Detergents n-dodecyl-maltoside (DDM), Lauryl Maltose Neopentyl Glycol (LMNG) and amphipol A8-35 were from Anatrace.
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DHPC, DPC, 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LMPG), 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LPPG), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (POPS) were purchased from Avanti Polar Lipids (Alabaster, AL). 2,2-didecylpropane-1,3-bis-β-D-maltopyranoside (MNG-3) and Amphipol A8–35 were purchased from Anatrace (Maumee, OH). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and N-lauroylsarcosine sodium salt (sarkosyl) were purchased from Sigma-Aldrich (St Louis, MO). Sodium dodecyl sulphate (SDS) was purchased from Avantor Performance Materials (Deventer, NL). Dihexanoyl-d22-phosphatidylcholine-d9 (d9DHPC) was purchased from FB Reagents (Boston, MA).
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All lipids used in our experiments were obtained from Avanti Polar Lipids. n-Decyl-β-d-maltopyranoside (DM), Triton X-100, and amphipol A8-35 were purchased from Anatrace. Bovine serum albumin (BSA), CF, and Tween 80 were obtained from Sigma-Aldrich.
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For exchanging the detergent LMNG with amphipol, bd-II was supplemented with a threefold mass excess of amphipol A8-35 (Anatrace, Maumee, OH, USA). The sample was incubated at 4 °C for 2 h while stirring. Excess detergent was removed by an addition of a 40-fold mass excess BioBeads (SM-2 resin, BioRad, Feldkirchen, Germany) and further incubation of 3 h at 4 °C under stirring. Excess amphipols were removed by size exclusion chromatography (Superose 6 Increase 10/300 GL, GE Healthcare), equilibrated in 20 mM MOPS, 20 mM NaCl, pH 7.0. Peak fractions were pooled and used for further analysis. For cryo-EM the samples were supplemented with 1 μM aurachin D prior to placing them onto the cryo grids.
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