Basic Protocol 1: Preparing Small Unilamellar Vesicles (SUVs) by Extrusion Materials -
Stock lipid solutions in chloroform or a 2:1 (v/v) chloroform:methanol mixture, at a concentration of 0.1 to 10 mg/ml -
Chloroform (ACS grade and above) -
Nitrogen gas (industrial grade or better; if from a central supply, use a hydrophobic filter to remove oils from the gas) -
1 to 4 ml rehydration solution [deionized (DI) water or TBS or PBS; see reciperecipes; see for selection] -
Deionized water (resistivity ≥18 MΩ and apparent pH 5.5) -
Argon -
25‐ to 50‐ml glass round‐bottomed flask (cleaned by piranha or base etching; see protocol 4 and protocol 5) -
Positive‐displacement pipet with capillary piston made of pure polypropylene (Gilson), or Hamilton syringes -
Rotary evaporator attached to a vacuum pump -
40° to 50°C water bath -
Benchtop vortex mixer -
Extruder: Lipex extruder (Northern Lipids) or Avanti Mini‐Extruder (Avanti Polar Lipids) NOTE: All stock lipid solutions should be stored in glass vials with Teflon caps or Teflon septa at −20°C or lower. Chloroformand methanol should be ACS grade or above. Alternate Protocol 1: Preparing SUVs by Probe Sonication Materials -
Isopropanol (ACS grade and above) -
Cleaning solution: 1:1 (v/v) isopropanol/water -
Deionized (DI) water (resistivity ≥18 MΩ and apparent pH 5.5) -
1 to 4 ml of lipid suspension (see protocol 1, steps 1 to 8) -
Nitrogen gas (industrial grade or better; if from a central supply, use a hydrophobic filter to remove oils from the gas) -
Argon -
Emery sheet (3/0 grit or finer) -
Ultrasonic processor equipped with a double‐stepped microtip (e.g., VCX750, Sonics & Materials) in a sound‐abating enclosure -
Ice bath -
Centrifuge that can reach 16,000 × g -
Microcentrifuge tubes Alternate Protocol 2: Preparing SUVs by Freeze‐ThawingBasic Protocol 2: Preparing Membrane Supports by Piranha Etching Materials -
1:1 (v/v) isopropanol/water -
Deionized water (resistivity ≥18 MΩ and apparent pH 5.5) -
Sulfuric acid (H 2SO 4; ACS grade) -
30% hydrogen peroxide (H 2O 2; ACS grade) -
Glass substrates Alternate Protocol 3: Cleaning Substrates by Base EtchingAlternate Protocol 4: Cleaning Substrates with Air/Oxygen Plasma Materials -
Nitrogen gas (industrial grade or higher; if from a central supply, use a hydrophobic filter to remove oils from the gas) -
Deionized water (resistivity ≥18 MΩ and apparent pH 5.5) -
Plasma generator (SPI Plasma‐Prep II, SPI Supplies/Structure Probe) -
Mechanical vacuum pump with oil filters (Leybold Vacuum Pumps, SPI Supplies/Structure Probe) -
Oxygen gas with regulator, optional Alternate Protocol 5: Cleaning Substrates with Ultraviolet Light/Ozone Materials -
Nitrogen gas (industrial grade or higher; if from a central supply, use a hydrophobic filter to remove oils from the gas) -
Deionized water (resistivity ≥18 MΩ and apparent pH 5.5) Support Protocol 1: Preparing Substrates with Diffusion Barriers (Gridded Substrates) Materials -
Hexamethyldisilazane (HMDS; ACS grade or higher) -
Photoresist (S1805 positive g‐line photoresist, Microchem) -
Photoresist developer (MicroDev; Microchem) -
Photoresist stripper (acetone, ACS grade or higher) -
Deionized water (DI water; resistivity ≥18 MΩ and apparent pH 5.5) -
Nitrogen gas -
Isopropanol (ACS grade or higher) -
General cleanroom (class 100 or above) -
Spin coater (Laurell Technologies) -
90°C hot plate -
Mask aligner (NXQ 4006, Neutronix‐Quintel) -
Photomask (design of the mask, usually 1‐ to 3‐µm features, can be generated by L‐edit Pro; Tanner EDA; quartz masks with the designed feature, such as parallel‐line grid patterns, are then manufactured by a mask‐making vendor) -
Metal target (99.99% chromium, Alfa Aesar) -
Thin film evaporator (Edwards EB3 electron beam evaporator; Edwards) Support Protocol 2: Preparing Substrates with Curvature Modulation Materials -
Hexamethyldisilazane (HMDS; ACS grade or higher) -
Photoresist (S1805 positive g‐line photoresist; Microchip) -
Photoresist developer (MicroDev, Microchem) -
Distilled water (DI water; resistivity ≥18 MΩ and apparent pH 5.5) -
Nitrogen gas -
Piranha solution (see protocol 4) -
5:1 buffered hydrofluoric acid (buffered oxide etch, 5:1 CMOS; Mallinckrodt Baker) -
Photoresist stripper (Acetone, ACS grade or higher) -
General cleanroom facility (class 100 or higher) -
Spin coater -
Hotplate -
Mask aligner (NXQ 4006, Neutronix‐Quintel) -
Photomask (design of the mask, usually 1 to 3 micron features, can be generated by L‐edit Pro; Tanner EDA; quartz masks with the designed feature, such as parallel lines, are then manufactured by a mask‐making vendor) -
Plasma etcher for SiO 2 anisotropic etching (AutoEtch 590, Lam Research) Basic Protocol 3: Formation and Functionalization of Supported Membranes Materials -
Nitrogen gas (industrial grade or higher; if from a central supply, use a hydrophobic filter to remove oils from the gas) -
Clean substrates ( protocol 4, Alternate Protocols protocol 53, protocol 64, and protocol 75) -
SUV suspension ( protocol 1, Alternate Protocols protocol 21 and protocol 32) -
Spreading buffer (2× PBS or 2× TBS; see reciperecipes; see for selection) -
Working buffer: deionized water or TBS (see recipe) or phosphate‐buffered saline (PBS; see recipe) -
Blocking solution: 5 mg/ml casein in PBS (see recipe for PBS) or 0.01% (w/v) bovine serum albumin (BSA) in PBS (see recipe for PBS) -
Aluminum foil, optional Support Protocol 3: Formation of Supported Membranes on Silica Beads Materials -
Spreading buffer: 2× TBS or 2× PBS (see reciperecipes) -
SUV suspension ( protocol 1, Alternate Protocols protocol 21 and protocol 32) -
Silica beads at 10 wt% solids in deionized water (Bangs Laboratories) -
Working buffer: deionized water or TBS (see recipe) or phosphate‐buffered saline (PBS; see recipe) Support Protocol 4: Formation of Supported Intermembrane Junctions Materials -
0.5 M sucrose, warmed to 50°C -
Dry lipid film in a round‐bottomed flask ( protocol 1, steps 1 to 6) -
Supported membrane (see protocol 10) -
Working buffer: DI water or TBS (see recipe) or PBS (see recipe) Basic Protocol 4: Characterizing Supported Membranes Materials -
Fluorescent samples (i.e., supported membrane with or without protein functionalization; see protocol 10) -
Standard supported membranes with known fluorophore concentrations (e.g., DOPC bilayers containing either BODIPY‐FL‐DHPE or Texas Red‐DHPE) -
Fluorescence microscope equipped with: -
Filter sets that match the excitation and emission spectrum of the fluorophore used -
Light source (typically a mercury lamp or a xenon lamp) -
Adjustable field diaphragm -
60× or higher objective lens -
CCD camera Support Protocol 5: Measuring the Scaling Factor Materials -
Standard SUV suspensions with known fluorescent lipid molarity (see protocol 1 for SUV suspensions and protocol 13 for selection of standard fluorophore) -
Fluorescent protein solutions with known protein concentrations -
Buffer (same as that used to make the fluorescent protein solution) |
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