Application Note

Channel:Cells:
Tools:Ompf
GUVs
Port-a-Patch

Lipid Bilayer recordings of OmpF
reconstituted in ProteoliposomesTheelectrophysiology

teamatNanion

Technologies

GmbH,Munich.

OmpF was kindly provided by Helge Weingart, Group of Prof Winterhalter, Jacobs
University Bremen.
Summary
Solvent-free planar lipid bilayers were formed in anThe mixture of GUVs and protein was incubated for 1 hour

automatic manner by bursting of giant unilamellar vesicles

at room temperature, followed by the addition of Bio-(GUVs) positioned by suction on the apertures of our patchBeads® SM-2 (Bio-Rad) at 40 mg/ml in GUVs solution. Theclamp chips made from borosilicate glass substrate. Incu-mixture was incubated with the Bio-Beads for 1 hour atbation of GUVs with purifi ed ion channel protein of interest

room temperature and then over-night at 4°C to remove

yielded proteoliposomes. These proteoliposomes allow for

the detergent. Bio-Beads were discarded after centrifu-

immediate recording of channel activity after GUV seal-

gation and the protein containing GUVs could be useding. This approach reduces the time consuming, laborious

immediately. When kept at 4°C, storage of the proteoli-and sometimes diffi cult protein reconstitution processes

posomes was possible for weeks in a standard Eppendorf

normally performed after bilayer formation.

cup in 1 M sorbitol.

Results
For formation of a planar lipid bilayer containing theOmpF, 1 to 3 µl of the proteoliposomes solution waspipetted onto the patch clamp chip. The microstructuredchip, which is commonly used in patch clamp experiments with cells, contains an aperture approximately 1 µmin diameter. The GUVs were positioned onto the aperturein the chip by application of a slight negative pressure.
Typically, -10 to -40 mbar were suffi cient for reliable positioning within a few seconds after GUV addition. When
the GUVs touch the glass surface of the chip, they burst
and form planar bilayers with a seal resistance of tens tohundreds of GΩ (Table 1). A schematic of the bilayer formation process is shown in Figure 1.
In contrast to conventional bilayer recordings, where reconstitution of proteins is achieved by adding the protein
after bilayer formation in the presence of detergent, weinsert the porin into the GUVs directly after the electroformation.

Download more Application Notes from www.nanion.de

Figure 1: The formation of a planar lipid bilayer by vesicle fusion onto a
microperforated glass chip is schematically shown.

Application Note
Table 1: Distribution of seal resistances of lipid bilayers formed with DPhPC in 10 %cholesterol GUVs. (n=161).

In conclusion, we have demonstrated that the Port-a-Patch
platform enables automated formation of planar lipid bilayers from lipid vesicles. The use of proteoliposomes for bilayer formation allows for direct recordings from the proteinof inerest without need for further reconstitution steps.

Figure 2: (a) Single channel opening of OmpF channel in 1 M KCl at atransmembrane potential of +50 mV. (b) Current–amplitude frequency histogram
constructed from 30 sec of single-channel data shown above.
The main open channel conductance level is 4,01 nS. (c) I–V relation of single
OmpF channels. (d) Typical recordings of ionic currents through a single trimeric
OmpF channel at 200 mV.

Electrophysiology

References

Patch clamp experiments were performed with the Port-

1. M. Kreier, C. Farre, M. Beckler, M.George and N. Fertig.
2008 Rapid Screening of Membrane Protein Activity:
Electrophysiological Analysis of OmpF Reconstituted inProteoliposomes. Lab Chip, 2008, DOI: 10.1039/b713982a.

a-Patch, using borosilicate glass chips with an aperture
diameter of approximately 1 µm. Experiments were donein symmetric solutions of 1 M KCl, 10 mM HEPES at pH 5,4
and pH 7.

Methods
GUVs
Planar lipid bilayers were obtained from Giant Unilamellar
Vesicles (GUVs) prepared by using the electroformation
method in an indium tin oxide (ITO) coated glass chamberconnected to the Nanion Vescile Prep Pro setup.
Proteoliposomes Preparation
Purified OmpF (0,3 mg/ml) in solution containing the detergent Octyl-polyoxyethylene (Octyl-POE) (3 %) and 50
mM NaCl was reconstituted into GUVs by mixing the solution containing GUVs (300 µl) and OmpF with a final concentration of protein between 0.8 – 1.7 µg/ml and a final
concentration of detergent between 0.01% to 0.02% inthe GUVs solution.

Nanion Technologies GmbHphone + 49 89 219 095-0Ganghoferstr. 70A

fax80339 Munich, Germany

www.nanion.de • info@nanion.de+49 89 218997960