Two-dimensional microelectrophoresis in supported lipid bilayers

Stelzle, Martin; Miehlich, R.; Sackmann, E.

doi:10.1016/s0006-3495(92)81712-5

Cited by 114 publications

(165 citation statements)

References 8 publications

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“…18 are known except the differential force ( f 1 Ϫ f 2 ), which was obtained from the fit procedure. The net force acting on the charged component was found to be 8.9 Ϯ 0.4 ϫ 10 Ϫ17 N per molecule, which is consistent with the applied field strength of 10-15 V͞cm and a typical amount of electroosmotic drag (15). The total area fraction of negatively charged lipid in this profile is 13%, which is in relatively good agreement with the 15 mol% initial condition.…”

Section: Resultssupporting

confidence: 78%

“…Application of an electric field tangent to the membrane plane imparts a force on each molecule. It has been shown that this electrophoretic force is the result of an electrical force acting directly on the charged molecules and an electroosmotic drag, produced by frictional coupling to the bulk electroosmotic flow (14,15). Assuming the total force per unit area acting on component i, f i , is independent of lateral position in the membrane, the contribution to the chemical potential from the electric field is A mi f i r where r denotes the position along the direction of the field.…”

Section: ͪ1ͪ [4]mentioning

confidence: 99%

See 1 more Smart Citation

Electric field-induced reorganization of two-component supported bilayer membranes

Groves

Boxer

McConnell

1997

Proc. Natl. Acad. Sci. U.S.A.

110

135

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Application of electric fields tangent to the plane of a confined patch of f luid bilayer membrane can create lateral concentration gradients of the lipids. A thermodynamic model of this steady-state behavior is developed for binary systems and tested with experiments in supported lipid bilayers. The model uses Flory's approximation for the entropy of mixing and allows for effects arising when the components have different molecular areas. In the special case of equal area molecules the concentration gradient reduces to a Fermi-Dirac distribution. The theory is extended to include effects from charged molecules in the membrane. Calculations show that surface charge on the supporting substrate substantially screens electrostatic interactions within the membrane. It also is shown that concentration profiles can be affected by other intermolecular interactions such as clustering. Qualitative agreement with this prediction is provided by comparing phosphatidylserine-and cardiolipin-containing membranes.Electric fields can be used to reorganize molecules in fluid lipid bilayer membranes. In 1977 Poo and Robinson (1) observed that application of an electric field caused the membrane-bound protein, Con A, to congregate on one side of a living cell. The distribution of protein at steady-state results from a balance between field-induced drift and diffusion within the confined membrane area (2, 3). The shape of a field-induced concentration profile thus contains information about the forces affecting molecules in the membrane. However, the small size and complexity of native cell membranes hinders analysis of these profiles. Recently, electric fields have been used to reorganize and concentrate lipids and proteins in supported bilayers where quantitative analysis is greatly simplified (4, 5).Supported bilayers can be created by spontaneous fusion of unilamellar phospholipid vesicles with an appropriate substrate such as silica (6, 7). The resulting membrane is typically separated from the solid substrate by a thin (10 Å) layer of water (8-10) and retains many of the properties of free membranes, including lateral fluidity. The fluidity is macroscopically long-range with mobile components of both leaflets of the bilayer freely diffusing over the entire surface of the support. The diffusive mixing and flow of molecules within the membrane can be confined by imposing barriers on this lateral motion. Manually scratching the membrane-coated surface can effectively create such barriers. Alternatively, more precisely partitioned membranes have been formed by using pre-patterned substrates to impose structure onto the membrane (11). Application of electric fields tangent to the plane of supported membranes partitioned by either method causes molecules to reorganize within the confined membrane corrals. The size and geometry of these corrals can be tailored to allow precise analysis of the field-induced concentration profiles.In this work, a general description of the steady-state electric field-induced reorganization of ...

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Section: Resultssupporting

confidence: 78%

Section: ͪ1ͪ [4]mentioning

confidence: 99%

Electric field-induced reorganization of two-component supported bilayer membranes

Groves

Boxer

McConnell

1997

Proc. Natl. Acad. Sci. U.S.A.

110

135

View full text Add to dashboard Cite

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“…A diffusion coefficient can be calculated from the images taken during the relaxation by spatial Fourier analysis of the fluorescence intensity distribution. The analysis was loosely based on the method outlined by Stelzle et al, 33 although the electrophoretic contribution was not included in our fitting. The fit was performed with the Levenberg-Marquardt algorithm.…”

Section: Resultsmentioning

confidence: 99%

Polymer-Supported Lipid Bilayers on Benzophenone-Modified Substrates

et al. 2000

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Solid-supported lipid membranes are important for their roles in fundamental biophysical research as well as in applications such as biosensors. In our study, lipopolymers containing alkyl side chains were synthesized and a mixture of the lipopolymer and free lipids was preorganized at the air-water interface and then transferred to a solid substrate using the Langmuir-Blodgett technique. A photochemical reaction between a substrate-functionalized benzophenone and C-H bonds on the lipopolymer was used to attach the lipopolymers to the substrate. The final assembly of the membrane was completed by vesicle fusion. Langmuir film experiments at the air-water interface indicate tighter molecular packing for the lipopolymers with 28 mol % alkyl side chains than for the ones with 22 mol %. Atomic force microscopy images point to phase separation of lipopolymers on the substrates due to their dewetting from hydrophobic surfaces. However, a mixture of lipopolymers and free lipids formed a smooth film on the same substrate. After the addition of the second lipid layer on the lipopolymer/free lipid layer, the fluorescence images of the polymer-supported bilayer suggested that the distal lipid layer is homogeneous on the micrometer scale. The relaxation of the fluorescent probe lipids was analyzed after application of an electric field to determine their diffusion coefficient; the distal lipid layer was mobile with an average diffusion coefficient of ∼0.1 µm 2 /s. Moreover, the immobile fraction of the lipids in the distal layer was estimated to be around 15%.

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“…ATTO-labelled CymA was introduced into POPC liposomes by the method based on that of Carter et al 23 Lipid vesicles, octylglucoside (OG) and CymA-ATTO565 were mixed by inversion to final concentrations of 16.4 mg ml À1 lipid, 45 mM OG and 55 mg ml À1 labelled protein/ lipid respectively. After 15 min incubation on ice, the lipid/ protein mixture was rapidly diluted 200 fold with buffer A, precooled to 4 C, and centrifuged at 142,000g for 1 h to pellet the proteoliposomes. The proteoliposomes were re-suspended in the same volume of fresh cold buffer A and centrifuged at 142,000g for 1 h to pellet the proteoliposomes.…”

Section: Protein Reconstitutionmentioning

confidence: 99%

Manipulation and sorting of membrane proteins using patterned diffusion-aided ratchets with AC fields in supported lipid bilayers

et al. 2012

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We present ratchets capable of directing the movement of charged components within supported bilayer lipid membranes. These ratchets make use of asymmetrically patterned features and AC electric fields, and have been demonstrated to transport charged species such as lipids and transmembrane proteins between two reservoirs. Proteins were present in both orientations in the membrane, with only those with their extra-membranous domain orientated away from the glass substrate being mobile. Proteins in the mobile orientation were transported using these ratchets, thereby sorting the two orientations from one another, and creating an area of the membrane containing five times more protein in one orientation than the other.

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Two-dimensional microelectrophoresis in supported lipid bilayers

Cited by 114 publications

References 8 publications

Electric field-induced reorganization of two-component supported bilayer membranes

Electric field-induced reorganization of two-component supported bilayer membranes

Polymer-Supported Lipid Bilayers on Benzophenone-Modified Substrates

Manipulation and sorting of membrane proteins using patterned diffusion-aided ratchets with AC fields in supported lipid bilayers

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