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- Johansson Fast, Björn, 1986
(författare)
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Applications of hydrodynamic forces for membrane chromatography
- 2013
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Licentiatavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Membrane proteins are proteins that reside in the cell membrane. Due to their position in the cell membrane, the barrier between the cell interior and exterior, they are vital to cell signaling. Moreover, the presence of membrane proteins in the cell's signaling pathways make membrane proteins important drug targets. In fact, one of the most important drug target classes are the membrane residing G protein coupled receptors. Despite this apparent importance there is currently no good technique for purifying membrane proteins that does not also have a high risk of causing denaturation of the protein. Taking into account that the cell membrane and its constituents can be described as a two-dimensional fluid, combined with the fact that many chromatography techniques are two-dimensional systems, it may seem peculiar that membrane chromatography is not yet solved in a satisfactory way. The aim of this thesis is to contribute to improved membrane protein chromatography.Hydrodynamic forces have recently been used to induce lateral movement of membrane associated molecules, a principle which has been utilized to improve the efficiency of the accumulation and separation of membrane associated molecules situated in supported lipid bilayers (SLB). This was done by forming SLBs in microfluidic channels, and by further introducing a gold barrier situated on the floor of the microfluidic channel. The barrier restricts the SLB to the center of the microfluidic channel where the spatial variation of the hydrodynamic force is small. Thanks to the so obtained homogeneous force, it was possible to show that separation of cholera toxin B subunit based on the number of attachment points to the SLB was complete, with no material in between the different populations.Also, a method for label-free diffusivity measurements on a substrate that is sensitive to changes in the effective refractive index in a small volume above the substrate is described. The technique is based on local application of hydrodynamic forces, in a so called hydrodynamic trap, to locally accumulate proteins at a surface coverage significantly higher than the equilibrium of the system. The trap is subsequently turned off, allowing the accumulated proteins to diffuse out of the trap, allowing the diffusivity of the accumulated proteins can be studied. The latter method was shown to provide a unique possibility to compare the diffusivity of labeled and non-labeled proteins in otherwise identical systems. It was shown that the inclusion of fluorescent labels decreases the diffusivity by approximately 12%.
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| 2. |
- Johansson Fast, Björn, 1986, et al.
(författare)
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Hydrodynamic separation of proteins in supported lipid bilayers confined by gold barriers
- 2013
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Ingår i: Soft Matter. - : Royal Society of Chemistry (RSC). - 1744-6848 .- 1744-683X. ; 9:39, s. 9414-9419
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Tidskriftsartikel (refereegranskat)abstract
- Hydrodynamic drag forces generated by liquid flow above a supported lipid bilayer (SLB) can be used to induce lateral movement of molecules protruding from the SLB. Since the velocity of the individual molecules depends on their size and coupling to the lipid bilayer, these forces can also be used to enrich and separate different types of membrane-bound molecules. To improve and better quantify hydrodynamic-based molecular separation in SLBs, we formed the SLB on the floor of a microfluidic channel which was patterned with gold barriers that confined the lipid bilayer to a 100 mm wide strip in the center of a 300 mm wide microfluidic channel. This forces the SLB into a region of the channel where the spatial variation of the hydrodynamic forces is close to zero while at the same time preventing the SLB from creeping up on the PDMS sides of the channel, thus reducing the loss of material. We here use this approach to investigate the accumulation of (i) fluorescently labeled lipids and (ii) the protein complex cholera toxin B (CTB) and to compare how the accumulation and separation differ when having an infinite reservoir or only a spatially limited band of studied molecules in the SLB. In addition, we show how the method can be used for complete separation of different polyvalently bound fractions of CTB.
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