We demonstrate a convenient chip platform for the addressable immobilization of protein-loaded vesicles on a microarray for parallelized, high-throughput analysis of lipid-protein systems. Self-sorting of the vesicles on the microarray was achieved through DNA bar coding of the vesicles and their hybridization to complementary strands, which are preimmobilized in defined array positions on the chip. Imaging surface plasmon resonance in ellipsometric mode was used to monitor vesicle immobilization, protein tethering, protein-protein interactions, and chip regeneration. The immobilization strategy proved highly specific and stable and presents a mild method for the anchoring of vesicles to predefined areas of a surface, while unspecific adsorption to both noncomplementary regions and background areas is nonexistent or, alternatively, undetectable. Furthermore, histidine-tagged receptors have been stably and functionally immobilized via bis-nitrilotriacetic acid chelators already present in the vesicle membranes. It was discovered though that online loading of proteins to immobilized vesicles leads to cross contamination of previously loaded vesicles and that it was necessary to load the vesicles offline in order to obtain pure protein populations on the vesicles. We have used this cross-binding effect to our benefit by coimmobilizing two receptor subunits in different ratios on the vesicle surface and successfully demonstrated ternary complex formation with their ligand. This approach is suitable for mechanistic studies of complex multicomponent analyses involving membrane-bound systems.
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Research Article|
June 06 2008
Addressable adsorption of lipid vesicles and subsequent protein interaction studies
Goran Klenkar;
Goran Klenkar
1Division of Molecular Physics, Department of Physics, Chemistry and Biology,
Linköping University
, Linköping, SE-58183, Sweden
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Björn Brian;
Björn Brian
1Division of Molecular Physics, Department of Physics, Chemistry and Biology,
Linköping University
, Linköping, SE-58183, Sweden
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Thomas Ederth;
Thomas Ederth
1Division of Molecular Physics, Department of Physics, Chemistry and Biology,
Linköping University
, Linköping, SE-58183, Sweden
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Gudrun Stengel;
Gudrun Stengel
2Division of Biological Physics, Department of Applied Physics,
Chalmers University of Technology
, Göteborg, SE-41296, Sweden
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Fredrik Höök;
Fredrik Höök
2Division of Biological Physics, Department of Applied Physics,
Chalmers University of Technology
, Göteborg, SE-41296, Sweden
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Jacob Piehler;
Jacob Piehler
3Institute of Biochemistry,
Johann Wolfgang Goethe-University
, Max-von-Laue-Str. 9, Frankfurt, 60438, Germany
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Bo Liedberg
Bo Liedberg
a)
1Division of Molecular Physics, Department of Physics, Chemistry and Biology,
Linköping University
, Linköping, SE-58183, Sweden
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Goran Klenkar
1
Björn Brian
1
Thomas Ederth
1
Gudrun Stengel
2
Fredrik Höök
2
Jacob Piehler
3
Bo Liedberg
1,a)
1Division of Molecular Physics, Department of Physics, Chemistry and Biology,
Linköping University
, Linköping, SE-58183, Sweden
2Division of Biological Physics, Department of Applied Physics,
Chalmers University of Technology
, Göteborg, SE-41296, Sweden
3Institute of Biochemistry,
Johann Wolfgang Goethe-University
, Max-von-Laue-Str. 9, Frankfurt, 60438, Germany
a)
Author to whom correspondence should be addressed; electronic mail: [email protected]
Biointerphases 3, 29–37 (2008)
Article history
Received:
March 13 2008
Accepted:
April 15 2008
Citation
Goran Klenkar, Björn Brian, Thomas Ederth, Gudrun Stengel, Fredrik Höök, Jacob Piehler, Bo Liedberg; Addressable adsorption of lipid vesicles and subsequent protein interaction studies. Biointerphases 1 June 2008; 3 (2): 29–37. https://doi.org/10.1116/1.2921867
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