Microarray Technology for Kinetic Analysis of Vesicle Bound Receptor-Ligand Interactions
Independent thesis Basic level (professional degree), 20 points / 30 hpStudent thesis
A proof-of-concept for a novel microarray used to study protein-ligand interaction in real-time using label-free detection is presented. Many of todays commercially available instruments lack the ability to immobilize membrane proteins. At the same time, the pharmaceutical industry develops drugs directed towards membrane-bound receptors. The need to study drug-target kinetics and to be able to screen for new medical substances is high. To study the biomolecular interactions in real-time, imaging surface plasmon resonance (iSPR) is used. A patterned sensor surface with hydrophobic barriers assisting in the piezodispensing of NeutrAvidin with complex-bound biotin-ssDNA is created. Histidine-tagged proteins are immobilized at the vesicle surface using divalent nitrilotriacetic acid. The concept of the vesicle immobilization, the protein-binding to vesicles and the protein-ligand interaction is initially studied using a Biacore instrument. The dissociation of the ligand IFNα2 from its receptor ifnar-2 (wt) are in accordance with the literature. In the imaging SPR experiments, it is found that the dissociation of IFNα2 from the ifnar-2 (wt) receptor is slower than expected, probably due to rebinding of the ligand. It is also found that imidazole is needed to avoid vesicle-vesicle interaction. The immobilization of proteins had to be done on-line i.e. when the vesicles were bound to the surface. Depending on the mixture of receptors at the vesicle surface the affinity for the ligand was changed. The results achieved were reproducible.
Place, publisher, year, edition, pages
Institutionen för fysik, kemi och biologi , 2007. , 64 p.
biosensor, SPR, vesicle, DNA
IdentifiersURN: urn:nbn:se:liu:diva-8739ISRN: LiTH-IFM-EX-07/1695-SEOAI: oai:DiVA.org:liu-8739DiVA: diva2:23432
2007-02-09, Planck, Fysikhuset, Linköpings universitet, 581 83, Linköping, 10:15
Klenkar, GoranEderth, Thomas