For two-dimensional (2D) crystallization experiments, the Ton subcomplex (ExbB/ExbD) was extracted and purified by IMAC as previously described. The sample was passed over a Superose 12 HR 10/30 column using 20 mM Tris-HCl, pH 7, 150 mM NaCl, 0.01% NaN3, and 0.035% Triton X-100. The purified complex was then mixed with a solution stock of E. coli polar lipid (Avanti Polar Lipids, Inc) at 10 mg/ml in 2% Triton X-100, to reach final concentrations of 0.5–1.0 mg/mL protein and 0.1 to 0.4 mg/mL lipid. The lipid-protein-detergent samples solution were placed into Mini Slide-A-Lyser dialysis devices (Pierce) with a 20 kDa MW cutoff, and dialyzed in 1 L of 25 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 0.01% NaN3 at 4°C. Aliquots of dialyzed samples were observed periodically by electron microscopy to monitor the formation of 2D crystals.
E coli polar lipid
Manufactured by Avanti Polar Lipids
Sourced in United States
E. coli polar lipids are a product derived from the cell membranes of the bacterium Escherichia coli. This product contains a mixture of polar lipids, including phospholipids and glycolipids, which are essential components of the bacterial cell membrane. E. coli polar lipids are commonly used in research and laboratory applications as a model system for studying membrane structure and function.
Automatically generated - may contain errors
Lab products found in correlation
4 one, by Cayman Chemical (2 mentions)
4 hne, by Cayman Chemical (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Sodium chloride (nacl), by Merck Group (2 mentions)
Hexane, by Merck Group (2 mentions)
Na2so4, by Merck Group (2 mentions)
Hexadecane, by Merck Group (2 mentions)
Nuclepore track etched membrane, by Cytiva (1 mentions)
Egfp antibody, by Roche (1 mentions)
Liss rhodamine pe, by Avanti Polar Lipids (1 mentions)
Imagequant las 4000 system, by Fujifilm (1 mentions)
Tls 55 rotor, by Beckman Coulter (1 mentions)
Glovebox, by Avanti Polar Lipids (1 mentions)
Membrane filter, by Cytiva (1 mentions)
N octyl β d glucoside β og, by Anatrace (1 mentions)
Bio beads sm 2, by Bio-Rad (1 mentions)
Nucleopore polycarbonate, by Cytiva (1 mentions)
Mini extruder, by Avanti Polar Lipids (1 mentions)
Sm2 bio beads, by Bio-Rad (1 mentions)
Infinite m1000, by Tecan (1 mentions)
15 protocols using e coli polar lipid
1
2D Crystallization of Ton Subcomplex
2
2D Crystallization of Ton Subcomplex
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For two-dimensional (2D) crystallization experiments, the Ton subcomplex (ExbB/ExbD) was extracted and purified by IMAC as previously described. The sample was passed over a Superose 12 HR 10/30 column using 20 mM Tris-HCl, pH 7, 150 mM NaCl, 0.01% NaN3, and 0.035% Triton X-100. The purified complex was then mixed with a solution stock of E. coli polar lipid (Avanti Polar Lipids, Inc) at 10 mg/ml in 2% Triton X-100, to reach final concentrations of 0.5–1.0 mg/mL protein and 0.1 to 0.4 mg/mL lipid. The lipid-protein-detergent samples solution were placed into Mini Slide-A-Lyser dialysis devices (Pierce) with a 20 kDa MW cutoff, and dialyzed in 1 L of 25 mM Tris-HCl, pH 7.0, 150 mM NaCl, and 0.01% NaN3 at 4°C. Aliquots of dialyzed samples were observed periodically by electron microscopy to monitor the formation of 2D crystals.
3
Reconstitution of Bacterial Membrane Proteins
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E. coli polar lipids (Avanti Polar Lipids, Inc.) were dried under N2, resuspended in pentane, and dried again. The dry lipids were suspended to a final concentration of 20 mg/ml in reconstitution buffer (buffer R): 300 mM KCl and 25 mM citric acid, adjusted to pH 7.0, with NaOH, to which 35 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate was added. The suspension was sonicated to clarity, and after a 1–2-h incubation, the purified protein was added to a 5-µg protein/milligram lipid ratio. Detergent was dialyzed out overnight in the desired buffer, and the resulting liposomes were fast frozen in ethanol/dry ice and stored at −80°C. For H+ transport, experiments in symmetrical Cl− liposomes were dialyzed in 0–1,000 mM KCl, 0–500 mM K2SO4, and 50 mM citric acid, pH 4.5, with [KCl] + 2[K2SO4] = 1,000. The external solution contained 1 mM KCl, 1 mM glutamic acid, 0–1,000 mM NaCl, and 0–500 mM Na2SO4, pH 4.5, with [NaCl] + 2[Na2SO4] = 1,000.
4
Liposome Flotation Assay for Protein Binding
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Liposome generation and flotation was performed as described in Vollmer et al., 2015 (link). In short, E. coli polar lipids (Avanti Polar Lipids) dissolved in chloroform and supplemented with 0.2 mol% 18:1 Liss Rhodamine PE (Avanti Polar Lipids) were vacuum-dried on a rotary evaporator, dissolved as liposomes in PBS by freeze/thawing cycles and extruded by passages through Nuclepore Track-Etched Membranes (Whatman) with defined pore sizes using an Avanti Mini-Extruder to generate small unilamellar liposomes of defined sizes. For liposome flotations, proteins (6 μM) were mixed 1:1 with liposomes (6 mg/mL) and floated for 2 hr at 55,000 rpm in a TLS-55 rotor (Beckman) at 25°C through a sucrose gradient. Binding efficiency was determined by Western blot analysis using an EGFP antibody (Roche, 11814460001, 1:2000). As secondary antibody, an anti-mouse, horseradish peroxidase conjugated antibody (Calbiochem, 401215, 1:5000) was used. The ImageQuant LAS-4000 system (Fuji) and the AIDA software were used to compare band intensities of start materials with floated liposome fraction.
5
Preparation of E. coli Polar Lipid SUVs
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E. coli polar lipids (Avanti Polar Lipids, Alabaster, AL, USA) in chloroform were dried under constant flow of nitrogen. After applying vacuum for 30 min, lipids were resuspended in SLB buffer (25 mM Tris-HCl pH 7.5, 150 mM KCl) to a final concentration of 4 mg/mL and incubated at 37°C for 1 h while vortexing every 20 min. Liposomes were then sonicated until clear and stored at– 20°C. Thawed SUVs were roughly 70 nm in average diameter, as determined by dynamic light scattering.
6
Purification and Reconstitution of PaNhaP
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The synthetic gene for wild-type PaNhaP (PaNhaPWT) was cloned with a C-terminal cysteine protease domain fusion into the pET21a plasmid. The PaNhaP mutation Glu-73 to Ala (PaNhaPE73A) was introduced by site-directed mutagenesis (19 (link)). The resulting plasmids were used to transform E. coli C41-(DE3) cells and target proteins were purified as described (5 (link)). Purified proteins were reconstituted at a lipid to protein ratio (w/w) of 4 into liposomes prepared from E. coli polar lipids (Avanti Polar Lipids, Inc., Alabaster, AL) as described (5 (link)).
7
Anaerobic Lipid Pull-Down Assay
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Lipid pull-down assays were performed anaerobically under a nitrogen atmosphere in an anaerobic chamber (Belle Technology Glovebox) as described previously using E. coli polar lipids (Avanti Polar Lipids) [26 (link)]. In these assays, 50 mM proline was used to reduce the flavin cofactor in the PutA proteins (0.3 mg/ml) during the incubation with lipid vesicles.
8
Reconstitution of Membrane Transporters
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50 mg/mL E.coli polar lipids (Avanti Polar Lipids, Inc.) were dissolved in chloroform and methanol mixture (3:1, v/v), the organic solvent was removed by nitrogen stream. The remained lipid layer was resuspended to 20 mg/mL in assay buffer containing 40 mmol/L MES, pH 6.5, 40 mmol/L KCl, and 2 mmol/L MgSO4. After fast frozen and thawed for 10 cycles, the mixture was extruded through 0.4 μm membrane filter (Whatman). 1% n-octyl-β-d -glucoside (β-OG, Anatrace), 20 mmol/L adenosine, and the protein hENT1 or GLUT3 (a glucose transporter) was then added into the mixture (protein: lipid = 1:100, w/w). For blank control, equal volume of the same buffer used in the final step of protein purification was added. After incubation at 4°C for 1 h, β-OG was removed by incubation with 320 mg/mL Bio-Beads SM2 (Bio-Rad) overnight. After separation from detergent-absorbed beads, the mixture was repeatedly frozen and thawed for five cycles. The proteoliposomes were extruded through 0.4 μm membrane filter again and collected by an ultracentrifugation step at 100,000 g for 1 h. The collected proteoliposomes were washed twice with the cold assay buffer. The proteoliposomes were on ice and resuspended in cold assay buffer before use with a final concentration of 100 mg/mL.
9
Lipid Bilayer Preparation and Characterization
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DOPC, DOPE, CL, NaCl, Na2SO4, MES, TRIS, EGTA, hexane and hexadecane were purchased from Sigma Aldrich GmbH (Germany). 4-ONE and 4-HNE were purchased from Cayman Chemical (Ann Arbor, MI, USA) or synthesized as previously described [16 (link)]. E. coli polar lipids were purchased from Avanti@ Polar Lipids, Inc. (Alabaster, AL, USA).
10
Liposome Preparation and Characterization
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Unilamellar liposomes were prepared at room temperature (≈23°C) by hydration of lipid films and extrusion through polycarbonate filters with 0.1‐μm pore diameter (Nucleopore Polycarbonate, Whatman) according to the method described by Avanti Polar Lipids (www.avantilipids.com ). The E. coli polar lipids and the extruding equipment used for liposome preparation were purchased from Avanti Polar Lipids. The liposomes were stored at −80°C in buffer solution (150 mM NaCl, 20 mM Hepes, pH 7.25). After incubation with 1 μM mM GSDMD and 0.2 μM caspase‐1 for 90 min at 37°C in buffer solution (50 mM NaCl, 100 mM Hepes, 5 mM TCEP, pH 7.4), the liposomes were adsorbed onto freshly cleaved mica in buffer solution (50 mM NaCl, 20 mM Hepes, pH 7.4) (Muller et al, 1997 ). After an adsorption time of 30 min, the sample was washed several times with the AFM imaging buffer (150 mM NaCl, 20 mM Hepes, pH 7.8) to remove weakly adsorbed protein. Buffer solutions were freshly made using nanopure water (18.2 MΩ cm−1) and pro‐analysis (> 98.5%) purity grade reagents from Sigma‐Aldrich and Merck. Each experimental condition characterized by AFM was reproduced at least three times. Liposomes made from E. coli polar lipids incubated in buffer solution but in the absence of GSDMD showed no arc‐, slit‐ or ring‐like structures when imaged by AFM (Mulvihill et al, 2015 ).
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