by Harald Zähringer Labtimes 03/2015
Researchers working on membrane proteins usually apply artificial lipid vesicles to study the interactions of membranes with proteins, peptides, drugs or other biomacromolecules. Artificial lipids are usually classified according to their size as small unilamellar vesicles (SUVs) with diameters of about 25 nanometres and large unilamellar vesicles (LUVs) having diameters larger than 100 nanometres. Both types are prepared by extrusion or sonication of multilamellar vesicles (MLVs).
Typical procedures to generate SUVs involve direct sonication using probe-tip sonicators or indirect sonication, applying bath or cup-horn sonicators. Due to the high-power densities of probe-tip sonicators, SUV formation is fast but also prone to lipid degradation, aerosol generation and contamination of the sample with metal ions stemming from the sonicator. Indirect sonication procedures on the other hand are slow and require extended sonication times. Moreover, both techniques may produce LUVs as a by-product that have to be removed in further purification steps.
Hence, Sandro Keller’s group at the University of Kaiserslautern, Germany, looked for a smarter way of SUV preparation and came up with a method that relies on a beaker resonator (Klingler et al., Anal. Biochem., 477, 10-12). At the heart of the beaker resonator is a beaker-shaped sonotrode that conducts the energy of the ultrasonic waves to the sample vial, placed inside the water-filled titanium beaker.
To produce SUVs, Keller’s team filled 500 to 1,000 μl of MLVs in a 5 ml flat-bottomed glass vial (diameter 2 cm) and placed the vial five to six millimetres above the water-filled beaker resonator. They filled the water just to the height of the lipid dispersion in the vial or slightly (1 mm) above and kept the temperature of the circulating water at 20° C.
The group sonicated different types of MLV samples (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine = POPC; 1,2-dilauroyl-sn-glycero-3-phosphocholine = DLPC; and an E. coli polar lipid extract) with the beaker resonator, applying maximal sonication power in intervals of ten seconds, which were interrupted by short rests (1 second). Subsequent size determinations, using dynamic light scattering, revealed diameters of 35 to 45 nanometres for SUVs generated from POPC and DLPC, and approx. 70 nanometres for SUVs resulting from E. coli lipid extracts. Larger vesicles were found in the vial only in trace amounts after sonication; additional characterisation of the sonicated POPC vesicles with 31P-NMR spectroscopy confirmed that the prepared SUVs were, indeed, unilamellar. Hence, producing SUVs with a beaker resonator seems to be a clever alternative to avoid downstream purification steps required in other techniques.
Last Changed: 25.05.2015