Twelve milligrams of total polar lipid extract (Avanti Polar Lipids, Alabaster) was dried under a stream of nitrogen, washed with methanol, and dried as before. Lipids were re-hydrated in 20 mM MES pH 6.5, 50 mM NaCl and, when required, compound was added to a final concentration of 20 mM from a 200 mM stock. Lipids were solubilised by vortexing in the presence of 1.5% (v/v) pentaethylene glycol monodecyl ether, and 50 μg protein was added. The samples were incubated on ice for 5 min, and pentaethylene glycol monodecyl was removed to form liposomes by addition of bio-beads (Bio-Rad, Hemel Hempstead, UK). Eight additions of bio-beads were made to the sample: the first four used 60 mg, and the final four used 120 mg. Between additions, the samples were incubated with inversion at 4˚C for 20 min. Following the final bio-bead addition, the samples were incubated overnight at 4˚C with inversion. bio-beads were removed by passage of the sample through empty micro-bio spin columns (Bio-Rad, Hemel Hempstead, UK). The external buffer was replaced with 20 mM MES pH 6.5, 50 mM NaCl using a PD10 desalting column (GE Healthcare, Little Chalfont, UK). When required, competitors were added externally to a final concentration of 20 mM.
Bio beads
Manufactured by Bio-Rad
Sourced in United States, United Kingdom, Germany
Bio-Beads are a type of chromatographic media used for protein purification and separation. They are made of a cross-linked polymer matrix and are available in a range of bead sizes and properties to suit various applications. Bio-Beads provide a high surface area for effective adsorption and desorption of target molecules.
Automatically generated - may contain errors
Lab products found in correlation
Superose 6 column, by GE Healthcare (14 mentions)
Amphipol a8 35, by Anatrace (7 mentions)
Biobeads, by GE Healthcare (5 mentions)
Superose 6 increase 10 300 gl column, by GE Healthcare (4 mentions)
Sodium cholate, by Anatrace (3 mentions)
Adenosine triphosphate (atp), by Merck Group (3 mentions)
Superdex 200 increase column, by GE Healthcare (3 mentions)
Vesicle prep pro, by Nanion Technologies (3 mentions)
Superdex 200 increase 10 300 gl column, by GE Healthcare (2 mentions)
Tris 2 carboxyethyl phosphine tcep, by Thermo Fisher Scientific (2 mentions)
Sodium cholate, by Bio-Rad (2 mentions)
Superdex 10 300 column, by GE Healthcare (2 mentions)
Dodecyl β d maltoside, by Merck Group (2 mentions)
1 2 diphytanoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (2 mentions)
Sodium meta arsenite, by Merck Group (2 mentions)
Luria broth, by Carl Roth (2 mentions)
Ni nta bead, by Qiagen (2 mentions)
Adenosine diphosphate (adp), by Merck Group (2 mentions)
Hepes, by Carl Roth (2 mentions)
Dodecyl maltopyranoside ddm, by Anatrace (2 mentions)
148 protocols using bio beads
1
Liposome Reconstitution and Supplementation
2
Reconstitution of EmrE Protein
3
Nanodisk Reconstitution Protocol
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Nanodisk reconstitution was performed as described previously (14 (link),37 (link),60 (link)). Briefly, purified and spin-labeled protein cysteine mutants were mixed with the membrane scaffold protein MSP1D1 (78 (link)) and presolubilized DMPC lipids in a 1:2:160 respective molar ratio. The mixture was diluted to 3–4 mL with lipid buffer (50 mM sodium phosphate (pH 7.5), 300 mM NaCl, 1% Triton X-100) and incubated at 25°C for 30 min. Then, 0.8–1 g of Biobeads (Bio-Rad Laboratories) were added per 1 mL of the mixture and left incubating with light agitation at 25°C for 4 h. Finally, Biobeads were removed and concentrated to a final volume of 35 μL. For PELDOR measurements, samples were diluted 1:1 with deuterated ethylene glycol, and 70 μL of the mixture was loaded in 3 mm (OD) quartz tubes and flash frozen in liquid N2.
4
Reconstitution of ABCG2 Transporter in Nanodiscs
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Membrane scaffold protein (MSP) 1D1 was expressed in E. coli strain BL21(DE3) and purified as previously described in ref. (42 (link)). A mixture of brain polar lipid (Avanti Polar Lipids) and CHS at a 4:1 ratio (w/w) was diluted to 10 mM with a buffer containing 20 mM HEPEs and 150 mM NaCl, pH 7.5. Lipids were solubilized with a 3x molar excess of sodium cholate using a sonic bath. Solubilized lipids were further incubated with detergent-purified ABCG2 and MSP1D1 at a molar ratio of 100:5:0.2 (lipid:MSP:ABCG2). Detergents were then removed by the addition of activated Bio-Beads (Bio-Rad) at 4 °C overnight. Bio-Beads were then removed by Econo-Pac Columns (Bio-Rad), and the flow-through was spun at 100,000g for 30 min (13 (link)). The supernatant was collected and concentrated before loading on a Superdex 200 Increase column equilibrated with buffer A. The peak fraction of ABCG2 nanodisc was collected for cryo-EM sample preparation, and purity was checked by SDS-PAGE.
5
Transfer of Amyloid-Beta Oligomers to Amphipols
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Three types of APols were used to transfer βPFOAβ42 from DPC to APols: A8-35, SAPols and NAPols. In each case, appropriate amounts of each APol, from a stock solution at 100 mg/mL prepared in water, were added to the βPFOAβ42/DPC sample to reach Aβ/APol ratios of 1:0.5, 1:1 and 1:2 (w/w). After addition of APol, the sample was gently shaken at 37°C for 20 min in a vortex allowing the formation of the βPFOAβ42/DPC/APol ternary complex. Vortex shaking was not intended to affect the morphology of the oligomer. Indeed, these are standard conditions extensively used in the literature to transfer membrane proteins from detergent conditions to APols (Zoonens et al., 2005 (link)). After vortex shaking, DPC was removed by adding Bio-Beads (Bio-Rad) at a 1:50 (w/w) DPC/Bio-Beads ratio. The samples were incubated at 4°C for 30 min on a wheel. Finally, Bio-Beads were removed by centrifugation. To determine the stability of the βPFOAβ42/APol complex after DPC removal, samples were analyzed immediately and also after 24 h incubation at 37°C.
6
Cryo-EM Sample Preparation for Env-Fab Complexes
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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.
7
ABCA4 Proteoliposome and Nanodisc Reconstitution
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The ABCA4 protein purified in W1 buffer was used for proteoliposome and nanodisc reconstitutions. Equal amounts of BPL and DOPE, both dissolved in chloroform, were mixed and dried under a nitrogen stream. The mixture was resuspended in W1 buffer to a final concentration of 5 mM. After sonication for 30 min and 10 freeze-thaw cycles, the BPL/DOPE (1:1, w/w) solution should be ready for use. Proteoliposome was prepared by mixing ABCA4 and BPL/DOPE (1:1, w/w) with a molar ratio of 1:3000. The detergent was removed by manually packed Sephadex G-50 (Sigma G5050) column pre-equilibrated with buffer R2 (50 mM Tris pH 7.5 and 150 mM NaCl). The proteoliposome fractions were manually collected for biochemical studies. Nanodisc was prepared by mixing ABCA4, MSP1E3D1, and BPL/DOPE (1:1, w/w) with a molar ratio of 1:10:1000, followed by detergent removal with Bio-beads (Bio-Rad). The reconstituted ABCA4 nanodisc was further purified by a Superose 6 Increase 10/300 GL column (GE Healthcare) equilibrated with buffer R2. The peak fractions were collected and concentrated for biochemical analyses.
8
Reconstitution of HMGCR-UBIAD1-BRIL Complexes
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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.
9
Purification of YaxA-YaxB Complex
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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.
10
Assembly of AdeJ into Nanodiscs
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To assemble AdeJ into nanodiscs, a mixture containing 20 μM AdeJ, 45 μM MSP (1E3D1), and 930 μM E. coli total extract lipid was incubated for 15 min at room temperature. Afterward, 0.8 mg/ml prewashed Bio-Beads (Bio-Rad) was added. The resultant mixture was incubated for 1 h on ice followed by overnight incubation at 4°C. The protein-nanodisc solution was filtered through a 0.22-μm nitrocellulose filter to remove the Bio-Beads. To separate free nanodiscs from AdeJ-loaded nanodiscs, the filtered protein-nanodisc solution was purified by passage through a Superose 6 column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 7.5) and 100 mM NaCl. Fractions corresponding to the size of the trimeric AdeJ-nanodisc complex were collected for cryo-EM.
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