Experiments quizThis is a short quiz about the experimental set-up of experiments. It will take only a couple of minutes to complete. Succes!Q 1: Double referencingWhy should you use double referencing? to correct for ligand versus reference differences to correct for blank (buffer only) injections to correct for bulk differences in the analyte to correct for drift during the analyte injection Q 2: Kinetic injectWhy should you use the 'Kinetic / high performance' inject method? to separate the sample from the flow buffer to have slow injections to have lower bulk shift jumps to have better dissociation curves Q 3: Slow dissociationWith a slow dissociation, the best set up is to use multi cycle kinetics single cycle titration kinetics short & long kinetics steady state kinetics Q 4: RandomizationRandomization of the samples is done because it minimizes systematic errors it can reveal systematic errors it proves the system is reproducible it minimizes carry over between samples Q 5: Kinetic estimationTo get a better estimation of the kinetics you repeat the method on the same sensor chip use a wide analyte concentration range add more analyte injections in the method repeat the method with different ligand and analyte batches Q 6: Mass transportHow do you detect mass transport limitation? by immobilizing less ligand by varying the flow rate during analyte injection by injection several analyte concentrations by varying the analyte injection time Q 7: Non specificWhat can you try to minimize non-specific binding? use a low pH running buffer use a higher flow rate lower the analyte concentration adding more salt or detergent to the running buffer Q 8: Baseline driftWhat can you do when there is baseline drift? use a higher flow rate immobilize less ligand flow for a longer period of time with flow buffer ad more salt or detergent to the running buffer Q 9: Concentration rangeWhat is the best analyte concentration range to inject? 0 – 1 µM 0 – 100% of Rmax 0.1 – 10 times the expected KD at least one concentration should saturate the ligand Q 10: ReplicateReplicate analyte injections are done to prove the system is stable and reproducible to have more curves to analyse to reveal dilution errors to minimize systematic errors Q 11: ReplicatesWhy should replicates be in separate capped vials? to minimize sample dilution to minimize sample evaporation to minimize carry over between samples to minimize sample loss to the vial wall Q 12: CalibrationSome SPR instruments use a calibration routine. Why? to initialyse the fluidics to get the best detector sensitivity to make sure the curves are separated well to make sure the system is equilibrated Q 13: StatementsWhich of the statements is not true? A slow dissociation needs a longer dissociation time than a fast dissociation to resolve the kinetics To determine the Rmax, the ligand has to be fully saturated SPR can be used for both high and low affinity interactions Both the association and dissociation curve follow an exponential curve Q 14: BuffersWhy should the buffers used be filtered and degassed? To prevent air spikes and drift To minimize the bulk effect To have a better buffer equilibration To have a better 1:1 interaction Q 15: KineticsTo determine the ka and kd of an interaction you need at least One analyte injection with 1x KD concentration + dissociation One analyte injection that is saturating the ligand + dissociation Two analyte injections to steady state + dissociation Two analyte injections with curvature + dissociation User Details Send me my results.Name: ( required)Email: ( required)