NanoBio: Preparation of Supported Bilayer
PREPARATION OF SUPPORTED LIPID BILAYERS
This procedure describes the assembly of supported lipid bilayers on a glass coverslip. The core of this procedure is placing a very clean, hydrophilic piece of glass on top of a concentrated solution of small unilamellar vesicles. Once the bilayer is formed, it must be protected from air.
Small unilamellar vesicles, extruded within ~ 1 wk. Glass coverslips, typically 18 x 18 mm #1.5 ICN 7x detergent, i.e. MP Biomedicals catalog # 097667093 Millipore water
Hot plate Ceramic coverslip holder, e.g. Thomas Scientific catalog # 8542E40 Oven capable of heating to 400C OR benchtop plasma cleaner OR piranha etch Crystallizing dish
Procedure: Preparation of Glass Substrates 1. Load coverslips into ceramic holder. Immerse in 20-30% 7x detergent (70-80% distilled water), which has been heated until it is clear, for 5 minutes. 2. Rinse coverslips thoroughly for 5 minutes with double distilled water. 3. Dry the coverslips with N2(g). 4. Bake for 4 hours at 400ºC. Alternatively, dried coverslips can be cleaned in benchtop plasma cleaner (3 x 30 s). After baking or plasma treatment, the coverslips should be extremely hydrophilic, with a contact angle close to zero degrees. Store the coverslips in a clean covered glass dish.
Preparation of Supported Bilayers 1. For most consistent results, both coverslips and vesicle solution should be used within a week of preparation. The presence of precipitate in the vesicle solution can be an indication that vesicles will not form supported bilayers. 2. Dilute vesicles to 1 mg/mL in 20mM phosphate 50mM NaCl pH 7.2 (or other salt containing buffer of your choice). 3. Place 30uL drop of vesicle solution in the bottom of a crystallizing dish (not-too-recently cleaned is best so that the droplet does not spread). Place a clean coverslip on top of the vesicle solution. 4. Wait ~ 30 s to allow the lipid bilayer to form on the surface of the glass coverslip. 5. Gently fill the dish with Millipore water. Avoid introducing air bubbles in the vicinity of the supported bilayer on the glass coverslip. Using tweezers, gently shake the coverslip underwater to remove excess vesicles. 6. Keeping the bilayer underwater, lay a second (not cleaned) coverslip over the bilayer-containing coverslip to create a bilayer sandwich, where the bilayer is the mayo on the inside of the sandwich.
Critical Steps: 1. Again, cleanliness of all glass surfaces is of key importance.
2. A common difficulty is that when the clean glass coverslip is placed on the vesicle drop, a small amount of lipid solution comes into contact with the upper surface of the glass coverslip. This will result in fluorescent lipid residue on the top of the coverslip. In this case, it is easiest to re-make the bilayer.
Troubleshooting 1. If supported bilayers are incompletely formed, let the vesicles remain in contact with the surface longer (5 minutes) or increase the ionic strength and concentration of divalent cations in your buffer.
2. Absence of lipid bilayers can have multiple causes. Common ones are old vesicles or coverslips, insufficient ionic strength of the buffer, and lipid compositions which do not form supported bilayers.
3. The presence of tubular vesicles, intact stuck vesicles, etc., alongside a bilayer is most frequently a result of insufficient cleaning of the glass coverslips.
Notes: 1. Lipid composition. We have had success with many different lipid compositions. It can be difficult (or impossible) to form supported bilayers that are highly negatively charged, e.g. greater than 20% phosphatidylserine.
2. To form supported bilayers of lipids that are gel-phase at room temperature, the vesicles must be stored above the gel-phase transition temperature.
3. Buffer. Several buffers work well, especially Tris and PBS based buffers. These buffers most frequently contain a small amount of salt (~50mM) buffered at pH 7-8. See Cremer & Boxer J. Phys. Chem. B 1999 for additional guidelines.
Last updated: Caroline Ajo-Franklin 06 August 2007.