Surface plasmon resonance imaging systems, such as Flexchip from Biacore, are capable of monitoring hundreds of reaction spots simultaneously within a single flow cell. Interpreting the binding kinetics in a large-format flow cell presents a number of potential challenges, including accounting for mass transport effects and spot-to-spot sample depletion. We employed a combination of computer simulations and experimentation to characterize these effects across the spotted array and established that a simple two-compartment model may be used to accurately extract intrinsic rate constants from the array under mass transport-limited conditions. Using antibody systems, we demonstrate that the spot-to-spot variability in the binding kinetics was <9%. We also illustrate the advantage of globally fitting binding data from multiple spots within an array for a system that is mass transport limited.
[Show abstract][Hide abstract] ABSTRACT: The detailed understanding of the functions and mechanisms of the actin and microtubuli cytoskeleton depended, besides innovative methods in live cell imaging, on the purification and labeling of its constituents. This allowed researchers to quantitatively measure filament stability, the rates of filament turnover as well as the determination of the influence of cofactors on filament formation and structure. Septins form the least understood class of cytoskeletal structures in nearly all eukaryotic cells so far examined. In yeast, they comprise a family of proteins (Cdc3, Cdc10, Cdc11, Cdc12, Shs1) that form a co-polymeric, ring-like structure beneath the membrane. This ring serves as a template for the formation of a new bud neck and as a landing pat for proteins involved in polar growth and cytokinesis. Further progress in investigating the mechanisms of septin-structure formation and regulation is hampered by the lack of protocols to modify homogenous samples of purified septins with useful probes for in vitro biochemical studies.
We present a protocol for the purification and labeling of yeast septin rods. The four individual septin subunits were co-expressed in E.coli. One subunit of the septin polymer was expressed as SNAP tag fusion protein allowing for rapid and stoichiometric labeling with derivatized Benzylguanine (BG). To demonstrate the applicability of our approach, we introduced two different SNAP tag substrates: septin rods labeled with fluorescent BG compounds enabled us to monitor the formation of filaments by fluorescence microscopy whereas BG-biotin was used to couple septin rods to a sensor chip for quantitative surface plasmon resonance binding experiments. In a first application, we determined the affinity and the binding kinetics of the yeast protein Bni5 to the individually coupled septin rods. In a further application we could demonstrate that a once formed septin rod hardly exchange its subunits.
The herein introduced protocol of purifying SNAP tag modified septins from E. coli allowed us to derivatize the obtained septin rods with probes for the further in vitro characterization of this class of cytoskeletal elements. The availability of a very diverse set of SNAP tag substrates should open the way to investigate different aspects of septin biochemistry in mechanistic detail.
"Observation of the reflected light at different incidence angles reveals a dip in the reflectivity is observed when the SPR conditions are fulfilled. Commercial available SPR biosensors systems  have proven effective in detecting the presence of multiple analytes and in determining their concentration . However the price of such systems is typically too high to enable their use as a point of decision screening tool. "
[Show abstract][Hide abstract] ABSTRACT: The simultaneous detection of multiple biological analytes is achieved for the first time using a single optical fiber based surface plasmon resonance (SPR) biosensor. This is achieved by collecting the plasmonic wave re-scattered by a rough metallic coating deposited onto two separate sections (sensing regions) of a single multimode optical fiber. The results obtained showed that two gastric cancer biomarkers (apolipoprotein E and clusterin) are detected in clinically relevant concentrations each on a separate sensing region, simultaneously, something that cannot be done in traditional fiber based SPR biosensors that read the transmitted or reflected light to monitor the position of the resonance. While this multiplexing demonstration has been performed with two different biomarkers, it paves the way for the multiplexed detection of a larger number of biomarkers using a simple fiber optic based SPR sensor for point of decision diagnostics.
Sensors and Actuators B Chemical 07/2013; 5:454-458. DOI:10.1016/j.snb.2013.03.131 · 4.10 Impact Factor
"For kinetic titration, several analyte concentrations are injected (usually in increasing order) across the ligand surface in a single binding cycle . In the ligand array format, analyte at one concentration is flowed over a matrix of ligand spots within a single large flow cell . These formats consume less reagent material and require minimal scouting and so can decrease the time required for the experiment. "
[Show abstract][Hide abstract] ABSTRACT: To explore the variability in biosensor studies, 150 participants from 20 countries were given the same protein samples and asked to determine kinetic rate constants for the interaction. We chose a protein system that was amenable to analysis using different biosensor platforms as well as by users of different expertise levels. The two proteins (a 50-kDa Fab and a 60-kDa glutathione S-transferase [GST] antigen) form a relatively high-affinity complex, so participants needed to optimize several experimental parameters, including ligand immobilization and regeneration conditions as well as analyte concentrations and injection/dissociation times. Although most participants collected binding responses that could be fit to yield kinetic parameters, the quality of a few data sets could have been improved by optimizing the assay design. Once these outliers were removed, the average reported affinity across the remaining panel of participants was 620 pM with a standard deviation of 980 pM. These results demonstrate that when this biosensor assay was designed and executed appropriately, the reported rate constants were consistent, and independent of which protein was immobilized and which biosensor was used.
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