SLBs are formed according to previous protocols (31 (link), 38 (link)). Briefly, a 20-mM solution of lipids (99.5% Egg PC and 0.5% DSPE-PEG [2000]-biotin) in chloroform is dried under a nitrogen stream and left in a vacuum chamber for at least 1 h in order to evaporate the solvent. When the labeling of the lipids was necessary, a small amount (0.05%) of Texas Red DHPE (Avanti) was added to the mix. The resulting lipid film is then resuspended to 2 mM in phosphate-buffered saline (PBS) (pH 7.4), sonicated for 30 min, and then extruded 10 times (using an Avanti Mini Extruder with 10-mm filter supports and a 100-nm pore Whatman membrane) in order to obtain small unilamellar vesicles (SUVs). SUVs are then incubated for 10 min inside the observation chamber and washed with at least a 10-fold volume of PBS, then with a twofold volume of A Buffer. SUVs are stored on ice and used within a week.
Mini extruder
Manufactured by Cytiva
Sourced in United States
The Mini Extruder is a compact and versatile lab equipment designed for the extrusion of various materials, including polymers, ceramics, and metals. It features a small footprint and is suitable for research and development applications. The Mini Extruder allows for the controlled and consistent processing of materials in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Polycarbonate membrane filter, by Cytiva (2 mentions)
Sephadex g50 resin, by Merck Group (2 mentions)
200 nm polycarbonate membrane, by Cytiva (1 mentions)
Freeze dryer, by Labconco (1 mentions)
Uv 2401 pc spectrophotometer, by Shimadzu (1 mentions)
Nuclepore, by Cytiva (1 mentions)
Polycarbonate membrane, by Cytiva (1 mentions)
Poly 4glu tyr peptide, by Merck Group (1 mentions)
Nucleopore, by Cytiva (1 mentions)
Ethylene glycol tetraacetic acid (egta), by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by GoldBio (1 mentions)
Amyl acetate, by Electron Microscopy Sciences (1 mentions)
Hepes, by GoldBio (1 mentions)
Potassium chloride (kcl), by Thermo Fisher Scientific (1 mentions)
Norland 65, by Norland Products (1 mentions)
Plusone repel silane es, by GE Healthcare (1 mentions)
Mgcl2, by Thermo Fisher Scientific (1 mentions)
D biotin, by Avidity (1 mentions)
Nuclepore track etch membrane, by Cytiva (1 mentions)
Nano ns 90, by Malvern Panalytical (1 mentions)
12 protocols using mini extruder
1
Synthesis of Biotin-Labeled Small Unilamellar Vesicles
2
DMPC Vesicle Solubilization Kinetics
3
Interaction of Plant Polyphenols with Lipid Bilayers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The interaction of plant polyphenols with lipid bilayers was studied by fluorescence spectroscopy in LUV suspensions, which were prepared according to previously described methods94 (link). Briefly, the stock solution volume for the required final total lipid concentration was added to a vial, and the solvent evaporated with a mild, continuous flow of nitrogen, followed by overnight vacuum. Two identical samples were always prepared, with and without fluorophore, the latter to be used as blank. After hydration with buffer 10 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), pH 7.4, 150 mM NaCl, samples were submitted to vortex stirring and freeze-thaw cycles. LUV suspensions were formed by extrusion (Avanti Mini-extruder) at 60 °C, by forcing the multilamellar vesicle suspension 21 times through polycarbonate filters with 100 nm diameter pores (Nuclepore, Whatman) and left to reach equilibrium overnight. The probes were added from stock solutions in ethanol to an aliquot of freshly prepared LUVs and equilibrated overnight to ensure complete probe incorporation into the membrane49 (link),95 (link). The same volume of solvent (less than 1% v/v) was added the other aliquot of LUV suspension, to be used as blank.
4
Liposomal Calcein Encapsulation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
5mg total lipid with a ratio of 4:2:1 PC:PE:PS (Avanti polar lipids) in chloroform was dried under vacuum to give a solvent-free film. This was hydrated in 1ml of Calcein buffer (50mM calcein, 100mM NaCl, 10mM Na2HPO4 2mM KH2PO4) and freeze-thawed three times. The lipid suspension was then extruded 10 times through a 1μm polycarbonate membrane filter (Whatman) using a Mini Extruder (Avanti polar lipids). Un-encapsulated calcein was removed from the extruded suspension by size exclusion chromatography using sephadex G-50 resin (Sigma Aldrich), while PBS pH 7.5 (liposome buffer) was used as the aqueous phase buffer. Liposomes were stored at 4 °C and used within 1 week of creation.
5
Purification and Liposome Preparation of NME3-His
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NME3-His was expressed from BL21 after 3 h of 0.1 mM IPTG induction at 30°C. Proteins were purified with HisPur Ni-NTA Resin beads (Thermo Fisher Scientific) dialyzed into storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM Dithiothreitol). Dialyzed proteins were centrifuged at 20,640 × g for 30 min before storage at −80°C.
For liposome preparation, indicated lipid mixtures were dried, rehydrated in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, and subjected to a series of freeze–thaw cycles before extrusion through polycarbonate membranes (Whatman) with a pore size ranging from 50 to 1,000 nm using Avanti Mini-Extruder.
For liposome preparation, indicated lipid mixtures were dried, rehydrated in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, and subjected to a series of freeze–thaw cycles before extrusion through polycarbonate membranes (Whatman) with a pore size ranging from 50 to 1,000 nm using Avanti Mini-Extruder.
6
Preparation of DMPC-Cholesterol Liposomes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The procedure for preparing the liposomes is summarized in Figure 1 b. As shown in Table 1 , the DMPC:cholesterol ratio was 11:3 (g/g). The two components were dissolved in chloroform, and the final concentration of the mixture was 1 mg/mL. The flask was then connected to a Büchi R205 rotary evaporator (Flawil, Switzerland) equipped with a 40 °C water bath. The solvent was removed under vacuum to give a homogeneous lipid film that coated the flask’s wall, which was then kept under continuous vacuum for another 2 h to remove all residual chloroform. The lipid film was then hydrated with PBS by sonication in a water bath at 24 °C, which is the DMPC transition temperature. After 1.5 h, the hydrated lipid film formed a homogeneous dispersion of liposomes that were subsequently extruded at room temperature using a mini-extruder (Whatman, Inc., Clifton, NJ, USA) and 0.2-μm polycarbonate filters. The extruded liposome was stored overnight at 4 °C for future experiments.
7
Kinase Activity Assay with SUV Incubation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kinase activity was measured in vitro using a continuous enzyme-coupled reaction system performed at 30C as previously described83 (link). Reaction buffer contained 20 mM Tris (pH 7.5), 10 mM MgCl2, and 100 µM ATP. Poly-4Glu:Tyr peptide (Sigma-Aldrich) was used as the phosphorylation substrate at a concentration of 1 mg/ml. Small unilamellar vesicles (SUV) containing DOPC and DOGS-NTA-Ni lipids (Avanti Polar Lipids) in a buffer containing 20 mM Tris (pH 7.5) and 10 mM MgCl2 were produced by extrusion through a membrane containing 100 nm pores (Whatman) using a mini-Extruder apparatus. The total lipid concentration of 10X SUV stocks was fixed at 2 mg/ml with Ni–NTA-DGS lipid at 5 mol percent.
Compounds were diluted from DMSO stock solutions into either heated kinase buffer (25 mM Tris–HCl, pH7.5, 20 mM MgCl2) or sodium acetate solution (pH 4.0), before further dilution into kinase buffer. Diluted compounds were pre-incubated with kinase and SUV for ~ 30 min on ice before starting the reaction. Due to limited solubility of the compounds in aqueous buffers, the compounds may have partially adsorbed to the lipid micelles and hence the final concentration of available compound may be overestimated.
Compounds were diluted from DMSO stock solutions into either heated kinase buffer (25 mM Tris–HCl, pH7.5, 20 mM MgCl2) or sodium acetate solution (pH 4.0), before further dilution into kinase buffer. Diluted compounds were pre-incubated with kinase and SUV for ~ 30 min on ice before starting the reaction. Due to limited solubility of the compounds in aqueous buffers, the compounds may have partially adsorbed to the lipid micelles and hence the final concentration of available compound may be overestimated.
8
Liposome Preparation by Film Rehydration
9
Reconstitution of Microtubule Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GTP and paclitaxel (Taxol) were purchased from Cytoskeleton (Denver, CO). Guanosine-5′-[(α,β)-methyleno]triphosphate, sodium salt (GMPCPP), the nonhydrolyzable analog of GTP, was purchased from Jena Bioscience (Jena, Germany). Piperazine-N, N′-bis (2-ethanesulfonic acid) (PIPES), ATP, and glucose were purchased from Sigma (St. Louis, MO). HEPES and dithiothreitol (DTT) were purchased from GoldBio (St Louis, MO). EGTA, MgCl2, and KCl were purchased from Thermo Fisher Scientific (Waltham, MA). Sterile collodion (nitrocellulose) 2% in amyl acetate and amyl acetate were purchased from Electron Microscopy Sciences (Hatfield, PA). d-Biotin was purchased from Avidity (Aurora, CO). Chloroform solutions of 1,2-dioleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl), mini extruder, Nuclepore track-etch membrane (50 nm) from Whatman, and filter support disks 10 mm from Whatman were purchased from Avanti Polar Lipids (Alabaster, AL). High vacuum grease, silicone elastomer base (184 Sylgard), and the corresponding elastomer curing agent were manufactured by Dow Corning (Midland, MI). 2% Dimethyldichlorosilane in octamethylcyclooctasilane (PlusOne Repel-Silane ES) was purchased by GE Healthcare (Chicago, IL). Optical adhesive Norland 65 was purchased from Norland Products (Cranbury, NJ).
10
POPC/POPG Lipid Vesicle Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lipid films of POPC/POPG mixture at 7:3 molar ratio were obtained from lipids dissolved in chloroform:methanol (2:1) in round-bottom flasks. Organic solvent was removed by drying the lipid film under a N2 flux, and then, under vacuum overnight. Lipid films were then hydrated with CBP buffer containing either 25 mM calcein for leakage experiments or 150 mM NaF for CD or 150 mM NaCl for tryptophan quenching by acrylamide and submitted to an intense vortex. LUVs were obtained by extrusion using an Avanti Mini-Extruder (Alabaster, AL) and double-stacked polycarbonate membrane (Nuclepore Track-etch Membrane, Whatman) in two steps: firstly 6 and 11 times through polycarbonate membranes of 0.4 and 0.1 µm pore size, respectively. Dynamic light scattering measurements with a Zetasizer Nano NS-90 (Malvern Instruments, Worcestershire, U.K.) of pure lipid vesicle suspension showed an average vesicle radius of 66± 6 nm. Only fresh vesicle suspension were used in the experiments. For leakage experiments, gel filtration was used to remove the fluorescent dye outside the liposomes.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!