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molecular interaction with an affordable A simple and flexible system that’s easy for you to configure and modify with off-the-shelf SPR is a highly specific and sensitive technique based on a surface designed for molecular interaction between a ligand and analyte of interest. This figure represents a typical planar surface based on a self-assembled monolayer of polyethylene glycol (PEG) and carboxylic acid polyethylene glycol terminal alkanethiolates on gold. Sensor slides with a variety of surfaces are commercially available, or can be prepared from purchased or synthesized chemicals. 50 nM Gold Layer
Glass Slide
Flowcell The flowcell creates the reaction space and conditions for molecular interactions. Dimension variables affect velocity over the surface and surface area exposed. A large volume/surface area with average flow or no flow may be desirable for ligand fishing where the aim is to isolate a specific molecule from a mixture for further study e.g. mass spectrometry. Kinetic rate experiments require small volume, high velocity flow to avoid mass transport issues. Reichert offers several sizes of flow cells and a static cavity type. Additionally, the prism/flowcell interface design allows the simple attachment of a flowcell of your own design – e.g. to do electrochemistry and SPR. Software National Instruments LABview software is used to control the SR7000 and report/archive data. This open approach allows users to modify the program to their specific needs where desired. As an experiment is being run, the results are reported in real time to the computer screen. The figure is an example of this data. Data can be exported into Excel, Origin, GraphPad Prism or a number of other programs like CLAMP (Center for Biomolecular Interaction Analysis, Univ. of Utah) or WINSPALL (WINSPALL, Juergen Worm, MPIP Mainz) Pump A key criterion in pump selection is the type of experiments that will be run. Typically, a high quality syringe pump provides the lowest noise, most stable baselines and even flow for kinetic and equilibrium analysis. A peristaltic pump can be used as a lower cost option, to flow cleaning solutions, and to continually recirculate analytes for long time span equilibrium experiments.
Fluidics Designed to use low cost off the shelf HPLC valves and tubing. This allows for easy change over if contamination or damage occurs. Additionally, there is a range of options that allow for selection of the most appropriate materials and tube sizes for the biomolecules and chemicals to be used.
New Surface Plasmon Resonance Instrument: • Flexible: you design it to do your work • Response vs. time and reflectivity data • For kinetics (on, off, equilibrium), relative affinity, sequence recognition, concentration, ligand fishing • For method development…before running more • Extremely sensitive: Savitzky Golay Smoothed Data rms Noise = 0.45 µRIU = 0.33 RU = 3.3e-05 deg.
(Raw Data rms Noise = 0.97 µRIU = 0.71 RU = • Excellent baseline stability: Maximum drift 3.1 µRIU/hour [1 µRIU = 0.73 RU = 7.3e-05 Deg] • Given ready chemistry (slide with surface and analyte to test against it), the instrument can be up and producing data within an hour out of the box.
This system provides an easy-to-understand and easy-to-use platform for monitoring molecular interactions The easiest way to understand this process is to walk • Flow analyte with the binding partner or target molecule through an experiment. These major steps are: in it over the SAM. Examples of what can be done are • Form a self-assembled monolayer (SAM) on the gold flowing different concentrations of the binding molecule surface of the glass slide. This is usually done off line to determine rate constants and equilibrium binding of the instrument or purchased ready to use.
constants, “fishing” a molecule out of a soup of molecules • Immobilization of a ligand to the SAM. The ligand to capture it for additional analysis, or determining the is usually one half of the chosen binding pair concentration of a molecule in solution.
Protein Immobilization on the Sensor SurfaceProteins may be immobilized on the surface with wide varietyof chemistries used extensively in affinity chromatography. Thisresponse vs. time chart shows a typical protein immobilizationon a carboxyl sensor surface. The mixture of EDC/NHS is flowedover the surface to transform surface carboxyls to NHS esters.
Protein dissolved in a Sodium Actate Buffer is then injectedover the surface. An amine in the protein displaces the NHSester forming an amide bond with the surface. Ethanolamine is used to deactivate the unreacted NHS esters.
Kinetic DataThe SR7000 produces real time kinetic data of biomolecularbinding including Protein/Protein, Protein/DNA, DNA/DNA,Protein/Drug and Receptor/Ligand interactions. The Figureshows the time response of Human Serum Albumin binding to Mouse Anti-Human Serum Albumin covalently immobilizedon the sensor surface. The forward (association) and reverse(dissociation) reaction rates at various concentrations of HumanSerum Albumin are fit to a binding model – For Example 1: 1binding (A + B AB). The mathematical fits provide rate constants (kon, koff) and the thermodynamic equilibrium association and dissociation constants (KD KA).
SPR Resonance Minimum DataReflectivity Vs. Angle data showing the SPR reflectivity minimummay be downloaded from the SR7000. This data may be comparedto models of the reflectivity of optical multilayer systems basedon Fresnel calculations. The equations are used in predictingthe shift in the SPR angle i.e., the angle at which a minimum in reflectivity is observed in an SPR variable angle experiment.
Shifts are predicted by the thickness and refractive index of thinfilms on the SPR sensor surface. The curves show the change in the SPR reflectivity minimum before and after covalentimmobilization of 1.5 ng/mm2 Human Serum Albumin to a self-assembled monolayer on the gold sensor surface. This shiftcorresponds to a refractive index change of 2000 µRefractiveIndex Units (µRIU).
Small Molecule Binding demonstrates theSR7000’s “Sensitivity” and “Baseline Stability”The figures show Warfarin (MW 308.3 Da) binding to humanserum albumin immobilized on the sensor surface and a controlsurface. The response change of the control surface is due tothe mass effect of the Warfarin in solution. The binding ofWarfarin to Human Serum Albumin reaches equilibrium toofast to measure reaction rates.
Equilibrium DataThe flexibility of the SR7000’s fluidic system allows configurationof the instrument for extremely fast or very slow thermodynamicequilibrium experiments. Fast-on, fast-off reactions and injectionslasting an hour or more are possible by varying sample loop size,tubing length/size from the injector to flow cell and by varyingflow rates over a wide range. Long-term equilibrium experimentsusing limited sample are possible by recirculating analytes with aperistaltic pump. With the SR7000 it is possible to conduct theseexperiments over a wide temperature range. The chart shows a Langmuir fit of equilibrium data for warfarin(MW 308.3) binding to human serum albumin immobilized onthe sensor surface. SR7000 Baseline Stability and NoiseThe SR7000 Uses a 3700 Pixel ccd array to image the SPR reflectivityminimum. The large number of pixels coupled with proprietaryillumination control and image analysis algorithms translates tohigh resolution and low noise.
SR7000 Temperature ControlTemperature Range: 10 to 90 Degrees Centigrade, temperatureis held to +/- 0.015 Deg. C.
The SR7000’s sensor surface temperature is programmable overthe entire temperature range. Users may select starting andending temperatures, the time to go from the start temperatureto the end temperature, and the size and number or steps totake in getting from temperature A to Temperature B. Thechart below demonstrates this unique feature of the SR7000.
(0.01” I.D.Tubing)7.5 µL (0.005” I.D.Tubing) Catalog No.
Description
Includes instrument, control and data collection software,micro flow cell, static cavity sample cell Includes injection valve, solvent select valve, valve stand, all required tubing and fittings PEG/PEG COOH terminal alkanethiolate self-assembled monolayer on gold *The precision and accuracy of measurements are largely dependent upon operator skill, laboratory equipment, temperature control, the accuracy of calibration standards, chemical stability of sample, and the physical characteristics of the sample. A product demonstrationis suggested to validate the true precision and accuracy of laboratory equipment for specific samples. Due to a policy of continuous development, we reserve the right to change specifications without notice.
For more information,
contact Reichert Analytical Instruments.
Reichert, Inc.
Important Notice
For your own protection, only purchase your Tel: (716) 686-4500 • Fax: (716) 686-4555 Reichert Analytical Instruments from and have them serviced by an Authorized Reichert Distributor.
www.reichertai.com

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