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Proteoliposomes

Proteoliposomes are lipid bilayer vesicles that incorporate functional proteins, enabling the study of membrane-bound biological processes.
These synthetic systems mimic the structure and dynamics of cellular membranes, providing a platform to investigate protein-lipid interactions, transport mechanisms, and signaling pathways.
Researchers can leverage PubCompare.ai's AI-driven platform to quickly locate and compare the latest Proteoliposomes research protocols from literature, pre-prints, and patents, identifing the most effective methods to optimize their experiments.
Thsi intelligent comparisons make it easy to find the best products and optimize your Proteoliposomes research.

Most cited protocols related to «Proteoliposomes»

1
X-Ray Structures of LeuT Mutant-Fab Complexes
Cited 19 times
LeuT mutants were expressed as previously described10 (link), monoclonal antibodies and Fabs were generated by standard methods, and x-ray crystal structures of the Fab complexes were solved by molecular replacement. Final models were obtained by an iterative process of manual model building and refinement against X-ray diffraction data. The functional properties of LeuT mutants were examined using scintillation proximity binding assays and uptake or exchange assays with LeuT reconstituted into proteoliposomes.
Krishnamurthy H, & Gouaux E. (2012). X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature, 481(7382), 469-474.
Publication 2012
Biological Assay Monoclonal Antibodies proteoliposomes Radiography X-Ray Diffraction
2
Planar Bilayer Integration of Engineered Connector
Cited 18 times
A two-step approach was used to incorporate the connector into the planar bilayer lipid membrane (BLM). The first step was the preparation of unilamellar lipid vesicles containing the reengineered connector as described above. The next step was to fuse the extruded liposome into a planar BLM (Fig. 2i). The fluidity of the lipid bilayer was demonstrated by FRAP (Fluorescence Recovery After Photobleaching) (Fig. 2h). An excitation light was focused continuously on the bilayer to bleach the dye. The photobleached area appeared dark. But after the light was off, it gradually recovered due to the diffusion of the fluorescent lipid.
A standard BLM chamber (BCH-1A from Eastern Sci LLC) was utilized to form horizontal BLMs. A thin Teflon film with an aperture of 70–120 µm (TP-01 from Easter Sci LLC) or 180–250 µm (TP-02 from Easter Sci LLC) in diameter was used as a partition to separate the chamber into cis- (working volume 250 µL) and trans- (working volume 2.5 mL) compartments. After the aperture was pre-painted with 0.5 µL 3% (w/v) DPhPC n-decane solution twice to ensure the complete coating of the entire edge of the aperture, these compartments were filled with conducting buffers (5 mM Tris/pH 7.9, TMS, or 5 mM HEPES/pH 7.9, with varying concentration of NaCl or KCl).
Formation of the bilayer membrane on the partition is a key step for connector insertion into the bilayer (Fig. 2i). Considering all experiments, the occurrence of successful connector insertions was about 47–83%, which varied from person to person based on BLM experience and the quality of prepared proteoliposomes. So far, we have carried out a total of 280 separate BLM experiments in which successful connector insertions were found.
For single conductance measurements, the giant liposome/connector complex prepared earlier must be extruded using a polycarbonate membrane with pore size of 200 nm or 400 nm to generate small unilamellar liposomes. This liposome stock solution was further diluted by 10–20 fold for the BLM experiments before use. For insertion of connectors, 0.5–2 µL of the diluted liposome solution was loaded into the cis-chamber.
Conductance was measured in two ways: the first was derived at specific but constant holding potentials, and the second from the slope of the current trace induced by a scanning potential starting at −100 mV and ramping to 100 mV after incorporation of GP10 connector into the lipid membrane (Fig. 3f and 3g).
Wendell D., Jing P., Geng J., Subramaniam V., Lee T.J., Montemagno C, & Guo P. (2009). Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature nanotechnology, 4(11), 765-772.
Publication 2009
BLM protein, human Buffers decane Diffusion Gigantism HEPES Light Lipid Bilayers Lipids Liposomes Membrane Fluidity polycarbonate proteoliposomes Sodium Chloride Teflon Tissue, Membrane Tromethamine Unilamellar Liposomes Unilamellar Vesicles
3
Purification and Reconstitution of Membrane Proteins
Cited 17 times

Protocol full text hidden due to copyright restrictions

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Publication 2010
11-cis-Retinal Biological Assay Bos taurus Cells Chromatography, Affinity Dithionite Fatty Acids Fluorescence Glycolipids Homo sapiens Lipids Mannose Membrane Proteins Monoclonal Antibodies Phospholipids proteoliposomes Rhodopsin Rod Cell Outer Segment Rod Opsins SDS-PAGE Serum Albumin Triton X-100
4
Single-Molecule FRET Analysis of LeuT Mutants
Cited 14 times
LeuT mutants were expressed in E. coli, purified, and labeled on targeted engineered cysteines with Cy3 and Cy5 maleimide. The functional properties of the labeled constructs were determined by measuring Leu binding by scintillation proximity assay, and Ala transport was measured after reconstitution of the protein into proteoliposomes. The fluorescence properties of labeled proteins were studied to establish specific and efficient labeling and to establish that the observed FRET changes likely arise from inter-dye distance rather than photophysical phenomena. Various constructs were created, each with two cysteine residues strategically placed for labeling. Purified, labeled protein was immobilized onto a passivated-glass surface via a streptavidin-biotin linkage. Fluorescence data were acquired using a prism-based total internal reflection (TIR) microscope. Fluorescence resonance energy transfer (FRET) efficiency was calculated and analysis of fluorescence and FRET traces was achieved using automated analysis software developed for this application. The single molecule traces were analyzed for LeuT in the presence and absence of the substrates sodium and Leu, upon addition of the transport inhibitors clomipramine and octylglucoside, and in response to mutations of the extracellular vestibule as well as the network of intracellular residues proposed to stabilize the inward closed state. Molecular dynamics simulations of the protein immersed in an explicit membrane, solvated with water molecules, ions and ligands, were carried out and long equilibrations (totaling >500 ns) were run to assess conformational changes.
Zhao Y., Terry D., Shi L., Weinstein H., Blanchard S.C, & Javitch J.A. (2010). Single-molecule dynamics of gating in a neurotransmitter transporter homolog. Nature, 465(7295), 188-193.
Publication 2010
Biological Assay Biotin Clomipramine Cysteine Escherichia coli Fluorescence Fluorescence Resonance Energy Transfer inhibitors Ions Ligands maleimide Microscopy Mutation octyl glucoside prisma Proteins proteoliposomes Protoplasm Reflex Sodium Streptavidin Tissue, Membrane Vestibular Labyrinth
5
Planar Bilayer Integration of Engineered Connector
Cited 14 times
A two-step approach was used to incorporate the connector into the planar bilayer lipid membrane (BLM). The first step was the preparation of unilamellar lipid vesicles containing the reengineered connector as described above. The next step was to fuse the extruded liposome into a planar BLM (Fig. 2i). The fluidity of the lipid bilayer was demonstrated by FRAP (Fluorescence Recovery After Photobleaching) (Fig. 2h). An excitation light was focused continuously on the bilayer to bleach the dye. The photobleached area appeared dark. But after the light was off, it gradually recovered due to the diffusion of the fluorescent lipid.
A standard BLM chamber (BCH-1A from Eastern Sci LLC) was utilized to form horizontal BLMs. A thin Teflon film with an aperture of 70–120 µm (TP-01 from Easter Sci LLC) or 180–250 µm (TP-02 from Easter Sci LLC) in diameter was used as a partition to separate the chamber into cis- (working volume 250 µL) and trans- (working volume 2.5 mL) compartments. After the aperture was pre-painted with 0.5 µL 3% (w/v) DPhPC n-decane solution twice to ensure the complete coating of the entire edge of the aperture, these compartments were filled with conducting buffers (5 mM Tris/pH 7.9, TMS, or 5 mM HEPES/pH 7.9, with varying concentration of NaCl or KCl).
Formation of the bilayer membrane on the partition is a key step for connector insertion into the bilayer (Fig. 2i). Considering all experiments, the occurrence of successful connector insertions was about 47–83%, which varied from person to person based on BLM experience and the quality of prepared proteoliposomes. So far, we have carried out a total of 280 separate BLM experiments in which successful connector insertions were found.
For single conductance measurements, the giant liposome/connector complex prepared earlier must be extruded using a polycarbonate membrane with pore size of 200 nm or 400 nm to generate small unilamellar liposomes. This liposome stock solution was further diluted by 10–20 fold for the BLM experiments before use. For insertion of connectors, 0.5–2 µL of the diluted liposome solution was loaded into the cis-chamber.
Conductance was measured in two ways: the first was derived at specific but constant holding potentials, and the second from the slope of the current trace induced by a scanning potential starting at −100 mV and ramping to 100 mV after incorporation of GP10 connector into the lipid membrane (Fig. 3f and 3g).
Wendell D., Jing P., Geng J., Subramaniam V., Lee T.J., Montemagno C, & Guo P. (2009). Translocation of double stranded DNA through membrane adapted phi29 motor protein nanopore. Nature nanotechnology, 4(11), 765-772.
Publication 2009
BLM protein, human Buffers decane Diffusion Gigantism HEPES Light Lipid Bilayers Lipids Liposomes Membrane Fluidity polycarbonate proteoliposomes Sodium Chloride Teflon Tissue, Membrane Tromethamine Unilamellar Liposomes Unilamellar Vesicles

Most recents protocols related to «Proteoliposomes»

1
Reconstitution of NCX1.4 Proteoliposomes
For preparing the NCX1.4-reconstituted proteoliposomes, 50–100 µl of purified NCX1.4 protein (~0.5 mg/ml) was added to a lipid mix of POPE:POPG (Avanti Polar Lipids) at a ratio of 3:1 (w/w), while maintaining a protein-to-lipid ratio of 1:50–100 (w/w). After 30 min of incubation, detergent was removed by adding SM2 beads and with gentle agitation at 4 °C for 14–18 h. Finally, the reconstituted proteoliposomes were collected by the centrifugation of 1.5 ml Eppendorf tubes at 60,000 × g for 1 h, and pellets were suspended in a minimal volume of storage buffer (20 mM Tris-HCl, pH 7.5, and 100 mM CsCl). Flash-frozen aliquots of reconstituted proteoliposomes were stored at −80 °C until use. Before the assay of ion-flux activities, the proteoliposomes were briefly sonicated in a water-bath sonicator and loaded with 80–160 mM NaCl (or CsCl), and 0.5 mM EGTA at 35 °C for 2 h.
Giladi M., Fojtík L., Strauss T., Da’adoosh B., Hiller R., Man P, & Khananshvili D. (2024). Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX. Communications Biology, 7, 463.
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Publication 2024
2
Proteoliposome Preparation for Ypt7 and SNARE Reconstitution
Not available on PMC !
Proteoliposome preparation was as described (Orr and Wickner, 2022) . In brief, β-octylglucoside in methanol in a 2 ml glass vial was mixed with chloroform solutions of each lipid, delivered with Hamilton syringes, and the chloroform/methanol solvent was removed under a stream of nitrogen for 30 min at room temperature. Vials were then centrifuged in vacuo for 3 h in a speedvac to complete solvent removal. The lipid pellets were overlaid with 400 μl of 50 mM HEPES/NaOH, pH 7.4, 375 mM NaCl, 25% glycerol, 2.5 mM MgCl 2 , nutated at room temperature for 3 h to dissolve the pellet, and stored at -80°C. Aliquots of frozen lipid: detergent micellar solutions were thawed by nutation at room temperature for 30 min, then placed on ice and supplemented with 350 μl of purified Ypt7-tm and SNAREs in Rb150 (20 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 10% glycerol) with 1% β-octylglucoside and 250 μl of either Cy5-Streptavidin (for Ypt7/R proteoliposomes) or biotinylated phycoerythrin (for Ypt7/Q proteoliposomes). Unless otherwise noted, Ypt7-tm was added in a 1:8000 M ratio to lipid, Qc, where present, was added in a 1:2700 M ratio to lipid, and R, Qa, and Qb, where present, were added in a 1:16,000 M ratio to lipid. The higher level of Qc was used to drive more complete assembly of 3Q-SNARE complex. Each 1 ml mixed micellar solution was dialyzed in 12-mm flat width, 25 KDa MWCO dialysis tubing against 250 ml of Rb150 + 1 mM MgCl 2 with 1 gm of Bio-beads with stirring overnight at 4°C in the dark.
Orr A, & Wickner W. (2024). Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion. Molecular biology of the cell, 35(5).
Publication 2024
3
Reconstitution and Characterization of Aquaporins
Purified AQPs were reconstituted into proteoliposomes by mixing them with Escherichia coli lipids (Avanti Polar Lipids, Alabaster, USA) solubilized in 5% OG. The lipid-to-protein ratio (LPR) was set at 30, and the reconstitution was performed in 20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM dithiothreitol (DTT), and 0.03% NaN3 with a lipid concentration of 2 mg mL−1. The mixture was incubated with gentle mixing (10 min at RT). OG was removed with Bio-Beads (2 h of incubation). The reconstituted proteoliposomes were extruded 11 times through an extruder (Avanti Polar Lipids) using a 200-nm Whatman polycarbonate membrane. Control liposomes were made in the same manner without protein. The size and polydispersity index (PDI) were measured using dynamic light scattering (DLS) on a Malvern Zetasizer NanoZS instrument (Malvern, UK) at 25 °C (3 measurements of 13 runs). Immunoblotting against the 6xHis-tag was done to confirm the integrity of the proteins. To assess the functional characterization of both AQPs, the osmotic water permeability (Pf) was measured by stopped-flow spectrophotometry in a PiStar-180 Spectrometer at 20 °C (Applied Photophysics, Leatherhead, UK) [37 (link)]. Pf was computed according to the equation: Pf = kexp V0/Av Vw Cout, where kexp is the fitted exponential rate constant (Figure S2), V0 is the initial mean vesicle volume, Av is the mean vesicle surface area, Vw is the molar volume of water, and Cout is the external osmolarity. Measurements were performed at different points (0 h, 48 h, and 1 week) and at different storage temperatures (4 °C, RT, and 37 °C).
Yepes-Molina L., Teruel J.A., Johanson U, & Carvajal M. (2024). Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation. International Journal of Molecular Sciences, 25(4), 1987.
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Publication 2024
4
Liposome Preparation for Endosome Proteins
Liposome preparation was performed as described26 (link),27 (link),62 (link). Basic liposomes consist of 79.7% PC (L-α-phosphatidylcholine), 20% PS (L-α-phosphatidylserine) and, 0.3% Rhodamine-PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine rhodamine B sulfonyl ammonium salt) (molar ratios). Liposomes containing an endosome lipid composition contain 31% PC, 11% SM (N-stearoyl-D-erythro-sphingosylphosphorylcholine), 14% PE (L-α-phosphatidylethanolamine), 3% PS, 40% cholesterol, 1% PtdIns (L-α-phosphatidylinositol) and 0.3% Rhodamine-PE. When PtdIns(3)P was included, the PC concentration was reduced accordingly.
The protein-to-phospholipid molar ratio was adjusted to that of early endosomes24 (link),63 (link). Accordingly, for EE-SNARE PL, the protein-to-lipid ratio of syntaxin 13, vti1a, syntaxin 6, Vamp4 was 1:2,000, 1:10,000, 1:1,200, 1:15,400, respectively. For Rab5, we quantified Rab5 and syntaxin 13 in enriched early endosomes prepared from HeLa cells (see above) by quantitative Western blot analysis using purified proteins as a standard, yielding a molar ratio of syntaxin 13: Rab5 (1:0.11), resulting in 1:16,700 as the protein-to-lipid ratio of Rab5. For proteoliposomes containing only two SNAREs, a protein:phospholipid ratio of 1:2,000 was used for each SNARE protein, and for proteoliposomes containing only one SNARE, the ratio was 1:1,000. Finally, 4-LE-SNARE-PL, a ratio of 1:2,000 was used for each of the four SNARE proteins.
Koike S, & Jahn R. (2024). Rab GTPases and phosphoinositides fine-tune SNAREs dependent targeting specificity of intracellular vesicle traffic. Nature Communications, 15, 2508.
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Publication 2024
5
Reconstitution of Cgs Protein in Liposomes
E. coli polar lipids at a concentration of 10 mg ml−1 in 25 mM HEPES pH 7.5, 100 mM NaCl were extruded through a 100 nm filter to generate liposomes. Purified Cgs at a concentration of 2 mg ml−1 was added to liposomes destabilized by 0.3% DDM at a 1:2’000 protein:lipid molar ration. Detergent was then removed by two rounds of fresh Bio-Beads. Resulting proteoliposomes were loaded onto a Sephadex G50 column equilibrated with buffer 1 (25 mM Tris pH 7.6, 100 mM NaCl). Concentration of protein in the eluted fractions was measured using the Bradford method (protein assay dye purchased form Bio-Rad). Fractions containing the proteoliposomes were combined and adjusted to 0.2 mg ml−1 protein concentration. The incorporation of Cgs into proteoliposomes was confirmed by SDS-PAGE.
Sedzicki J., Ni D., Lehmann F., Stahlberg H, & Dehio C. (2024). Structure-function analysis of the cyclic β-1,2-glucan synthase from Agrobacterium tumefaciens. Nature Communications, 15, 1844.
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Publication 2024

Top products related to «Proteoliposomes»

1
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Bio-Beads SM-2 are macroporous polystyrene beads designed for size exclusion chromatography. They have a porous structure that allows for the separation of molecules based on their size and shape. The beads have a specified surface area and pore size distribution.
2
Bio beads by Bio-Rad
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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.
3
E coli polar lipid extract by Avanti Polar Lipids
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E. coli polar lipid extract is a laboratory product derived from the cell membranes of Escherichia coli bacteria. It contains a complex mixture of polar lipids, including phospholipids and glycolipids, which are the primary structural components of bacterial cell membranes. This extract is commonly used in research applications involving the study of membrane structure, function, and lipid-protein interactions.
4
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Axopatch 200b amplifier by Molecular Devices
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More about "Proteoliposomes"

Proteoliposomes are synthetic lipid bilayer vesicles that incorporate functional proteins, enabling the study of membrane-bound biological processes.
These systems mimic the structure and dynamics of cellular membranes, providing a platform to investigate protein-lipid interactions, transport mechanisms, and signaling pathways.
Researchers can leverage AI-driven tools like PubCompare.ai to quickly locate and compare the latest Proteoliposomes research protocols from literature, pre-prints, and patents, identifying the most effective methods to optimize their experiments.
Proteoliposomes are closely related to other membrane-based systems like Bio-Beads SM-2, Bio-Beads, E. coli polar lipid extract, and SM2 Bio-Beads, which are commonly used in membrane protein purification and reconstitution.
Similarly, Mini-extruder and LiposoFast are used for the preparation of Proteoliposomes.
Specialized equipment like the Axopatch 200B amplifier can be used to study the electrophysiological properties of Proteoliposomes, while data analysis tools like GraphPad Prism 7 can be employed to interpret the results.
When working with Proteoliposomes, researchers often use phospholipids like 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) to mimic the lipid composition of cellular membranes.
By incorporating membrane proteins into these synthetic systems, scientists can investigate their function, regulation, and interactions in a controlled environment, ultimately leading to a better understanding of cellular processes and the development of new therapeutic approaches.