The overall goal of this procedure is to prepare giant uni lamellar vesicles in a manner compatible with the functional integrity of membrane proteins incorporated into the vesicle membrane. This is accomplished by first depositing lipids or small liposomes onto a conductive surface like an ITO coated glass slide. The second step is to evaporate the solvent in a controlled manner to achieve a uniform lipid film on the conductive surface.
Next, carefully rehydrate the lipid film in a suitable buffer and prepare the electro formation chamber. The final step is to apply an oscillating voltage across the lipid film to generate giant unal Miller vesicles. Ultimately, the vesicles will be imaged to determine their size and quality and compare to vesicles formed using a classical technique.
Visual demonstration of this protocol is useful because lipid deposition can be difficult to master, depends on many variables and is best assay by visual inspection. Before starting this procedure, remove the lipids from storage at minus 20 degrees Celsius or minus 80 degrees Celsius and warm to room temperature. If possible, perform the experiment in an inert gas atmosphere such as a glove box.
Next, cover the lipids in dry argonne or nitrogen gas and in all steps minimize exposure to air. If the lipids are not supplied in organic solvent, suspend 0.3 milligrams of lipids in chloroform or cyclo heane at 10 milligrams per milliliter. Note that manufacturers stated concentrations are typically nominal only.
Use ethanol to clean both sides of 2 25 millimeter by 37.5 millimeter ITO coated glass slides. Then add 0.1 more percent Texas red DPPE to the lipids for fluorescent imaging. Measure the surface resistance with a multimeter to verify that the ITO coated side of the glass slides is facing up, which is indicated by a resistance of a few hundred ohms.
Aspirate the lipids into a solvent resistance syringe with the syringe needle not quite touching the ITO surface. Slowly apply the lipid by moving the needle back and forth across the slide. Cover the surface evenly, looking for a rainbow sheen on the glass surface.
Following this place the slides quickly into a vacuum desiccate and pump the chamber to less than one tor vacuum for 0.5 to one hours. To remove any trace solvent. Release the vacuum with inert gas.
Apply a thin layer of silicone grease to both sides of a silicone gasket and then place the gasket on the slide. Leave at least five millimeters of uncovered slide exposed at one end of the slide. Next, carefully hydrate each well by placing a 27 gauge syringe needle at the edge of the gasket and slowly apply buffer overfill each gasket well by approximately 10%with three millimolar heaps.
Once the lipids are hydrated, proceed quickly through the remaining steps. Since lipid films begin to delaminate, immediately verify the conductive side of the slide with the multimeter. Then apply the second ITO slide conductive face in to the top of the gasket.
Make sure to have at least five millimeters of overhang outside the gasket area and opposite the first overhang, and press gently to ensure a good seal. Clean the two overhangs using ethanol. Now secure the ITO slide sandwich to electrical contacts such as copper bars or gasket foam.
This graphic shows an exploded view of the chamber and contact assembly as a reference. Detailed plans can be found in the text protocol using a multimeter. Verify that there is no electrical short between the contacts and that the contacts connect properly to the ITO surfaces.
Next, place the electro formation chamber in an oven and heat the chamber 10 degrees Celsius above the highest melting temperature of any of the lipids present. Measure the gap between the two ITO coated surfaces to determine the amplitude of the sine wave. The amplitude of the sine wave should be approximately 0.7 volts RMS For each millimeter gap between the ITO surfaces, apply a 10 hertz sine wave for 60 to 90 minutes.
Connect the generator to the electro formation chamber and confirm the voltage with a multimeter. After 60 to 90 minutes of electro formation, giant unal vesicles should have formed. Use an inverted microscope equipped with a filter cube for Texas red dye to image the liposomes.
The liposome should be spherical, predominantly Ella by eye and free of defects such as strings hanging off the liposome. The preceding steps covered the classic preparation of giant liposomes from pure lipids. A more challenging preparation using small liposomes is shown.
Next first, prepare a saturated potassium carbonate solution of 44%relative humidity at 20 degrees Celsius. Use a plastic yogurt and granola container with a nesting upper cup that has been modified with holes in the upper cup. Place the saturated salt solution and excess salt in a tightly sealable container with an interior shelf.
Replace the shelf after filling and make sure the fluid is five to 10 millimeters below the shelf. Place a humidity sensor in a notch in the side of the container. Clean both sides of ITO coated glass slides with ethanol.
Use the multimeter to determine which side is conductive and label the non-conductive side with the sample name. Next, lightly grease both sides of a one or multiple hold silicone gasket. Place the slide's conductive, site up on the bench and apply the silicone gaskets to each slide to which lipid will be applied.
Making sure that there is at least five millimeters of exposed slide at one end to connect to the electro formation apparatus. Smooth the gasket to ensure a good seal, dilute the vesicle preparation in the low salt iso modic buffer to approximately one to two milligrams per milliliter. Apply the lipid to the slide in one to 10 microliter drops.
Place the slide on the interior shelf above the saturated salt solution and seal the container tightly. Leave at room temperature for three hours to overnight. The slide can now be used for the electro formation of giant liposomes as shown previously in the classic protocol.
Note the rainbow appearance of the individual deposits. This image shows liposomes electro formed from lipids deposited out of chloroform, the arrows point to giant liposomes. This image has very few fuzzy areas and thus indicates a good preparation.
This next image has many fuzzy regions and so indicates a generally poor preparation. Note that arrow two indicates a region with poorly distinguishable liposomes and that no liposomes can be brought into focus in the area indicated by arrow three. Shown here is a 10 x magnification of liposomes successfully electro formed from lipids deposited by dehydration of small liposomes.
The arrows indicate three good quality liposomes ranging in size from approximately five to 20 micrometers at 40 x magnification. A clear ring can be seen at the edge of the liposome, which indicates a uni or nearly lumal liposome. The capsaicin activated TR PV one ionic currents recorded in lipid membrane patches excised from the formed giant liposomes are pictured here.
The leak current indicated a 500 mega seal resistance before capsaicin was added. Saturating capsaicin activated a large T RRP V one current. The current return to near baseline as capsaicin is washed out of the patch While attempting this procedure.
It's important to remain flexible since many factors affect how the lipids dehydrate and form an even film on the glass substrate. Consult our online troubleshooting section and vary temperature, humidity, lipid concentration, and droplet size to achieve best results.
