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Multiple layer gradients flotation Schreiber et al [32] diluted OptiPrep with Krebs-Henseleit buffer containing 1.25 mM CaCl2 and 1.2 mM Na2SO4 to produce solutions of 17%, 11.5% and 8.4% (w/v) iodixanol

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For analysing the interaction affinity, the dissociation constant K d for each ligand was determined by analysing the normalised fluorescence (F norm []) versus ligand concentration using NanoTemper Affinity Analysis software and Prism Graphpad