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What is a label free screening system for therapeutic compounds? |
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It is a system to measure the interaction between proteins which cause diseases and compounds, without using labels such as fluorescence and RI. |
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What is SPR? |
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SPR is the abbreviation for Surface Plasmon Resonance. It is the technology to detect changes in refractive index near the surface of a gold thin film by examining the resonance pattern which forms between the free electron waves called plasmon forming on the surface of the gold thin film and the light coming into the gold film. A protein which causes a certain disease is immobilized on the gold film, various compound solutions are injected over it, and then some compounds bind to the protein, creating changes in the refractive index near the gold surface. Refractive index correlates with mass (density), and changes of the refractive index are shown as changes of the resonance pattern. By detecting the changes by CCD, compounds which are therapeutic candidates can be identified. Please refer to "Principles and Technology". |
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What are the major applications of AP-3000? |
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Please refer to the "Major Applications". AP-3000 can be used for far more applications than those listed here. Please contact us for information on other applications. |
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What are the features of AP-3000? |
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Please refer to the "Product". |
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How is AP-3000 structured? |
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AP-3000 is composed of an instrument and an external computer for analysis. Spotfire DecisionSite for AP-3000, AP-3000 Planner and other software are installed on this external computer. |
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What is the size of the instrument? |
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W:1.5m x L:0.8m x H:1.65m. For further information, please refer to the "Specification" section. |
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What is the weight of the instrument? |
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600Kg. Please refer to the "Specification" section. |
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What is the price of AP-3000? |
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Please contact us directly. |
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Can the temperature be altered? |
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Measurement can be taken only at the ambient temperature of 18-28 deg C. |
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What sensitivity does AP-3000 have? |
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With SPR based measurement, signals depend on molecular weight. AP-3000 is able to detect compounds with molecular weight of 100 Da. |
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How much is the running cost? |
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It depends on the measurement conditions. Please contact us for details. |
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What are the consumables specifically used with AP-3000? |
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Sensor sticks, Precise tips (Pipette tips), and reagents to immobilize proteins. |
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What are the features of the sensor? |
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Please refer to "Fundamental principle and Technology". |
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What is the price of a sensor stick? |
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Please contact us directly. |
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Are there any peripheral instruments required? |
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When preparing analyte solutions in a 384-well plate, a 384 (96) head injector, an 8-16 channel dispenser, a dielectric device for Plate, a plate centrifuge and a heat sealer are required. For biotinylation of a protein, a cooling centrifuge is required. For measuring concentration of a biotinylated protein, a micro spectrophotometer is good to have. |
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Can AP-3000 evaluate affinity? |
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Yes. The data mining software, Spotfire DecisionSite for AP-3000 enables automatic calculations of KD values. |
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Can AP-3000 evaluate kinetics? |
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Yes. It calculates kd, using dissociation curves. Contact us regarding further detailed calculations. |
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It is often thought that preparation for SPR measurements is not easy, and analysis of measurement results is difficult and takes a long time. Can high throughput measurements be truly done? |
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SPR based measurements call for preparation of plates with the exact volumes of analyte and buffer solutions. Because of this, it looks difficult to prepare plates, but if the optimized sample preparation method which is recommended is used, there should not be any problem. At the same time, using the customized software, Spotfire DesisionSite for AP-3000, the data analysis after screenings can be done promptly. Sometimes, any mistake in adjusting buffer solutions offsets signals (A phenomena in which all signals shift toward either plus or minus.). Even if this happens, data can be corrected by the Spotfire after the screening. |
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How much protein is used? |
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It depends on the types of proteins and immobilization methods, which are used. For instance, when carbonic anhydrase is immobilized by the Avidin-Biotin coupling method, one area of protein immobilization can be done with minimum of 0.5ug (100ug/mL x 5uL). It is possible to measure 400 points per one area at maximum, and therefore the amount of the protein used to measure one sample is merely 1.25ng. |
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How does the activity of proteins get measured? |
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AP-3000 has the function to recover injected solutions and this function is used to measure the protein activity. A substrate solution that enables functional assays is needed in order to measure the activity of proteins on AP-3000. The procedure to measure the activity is that after the substrate solution is injected and incubated in the flow-paths with an immobilized protein, the solution is recovered, and the changes occurred in the solution are measured by instruments other than AP-3000 (such as a plate reader). |
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My Proteins are unstable in the room temperature. Can we measure them anyways? |
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There is a refrigerated area within AP-3000, and proteins and some reagents can be kept in refrigeration (0-4 deg C) till the time of use. |
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Is it possible to re-use sensor sticks with immobilized proteins to measure compounds in order to determine measurement conditions? |
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Yes. Once a used sensor stick is returned to its case, it cannot be used again. (Such a sensor stick may or may not work, but it cannot be guaranteed.) At an experiment mode after immobilization of a protein, measurements of the compound binding and the protein activity can be taken at any desired order and number of measurements (only for 96 plates). At a screening mode, AP-3000 automatically inserts and disposes sensor sticks, and therefore they cannot be reused. |
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In what conditions are compounds set into AP-3000? |
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A regular screening is prepared in 384 well plates (maximum of 10 plates). It is also possible to do measurement with one 96 well-plate for a testing purpose. At either case, after preparing a plate, it is necessary to heat-seal it to prevent evaporation of the solutions. (Evaporation will cause signal deviation.) |
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Calibration is needed with SPR measurement, when DMSO is used. How does AP-3000 handle this problem? |
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Calibration is done on AP-3000. The calibration buffer with two DMSO concentration levels need to be prepared, which are different from the level of the running buffer. Using these buffer solutions, the instrument automatically performs measurement and calibration. The data taken before the calibration can be examined by the Spotfire. |
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Measurement of small molecules calls for DMSO. Can we use DMSO on AP-3000? What is the upper limit of concentration? |
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The maximum concentration of DMSO for the running buffer is 10%. |
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What is the upper limit of concentration for compounds? |
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Since the solubility of compounds depends on the kinds of compounds, there is no upper limit set for the compound concentration. Measurements have been taken with non-soluble materials. |
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With fragment screening, concentration of the compound is high, and this causes insolubility. It is difficult to confirm and eliminate any precipitation. Is it possible to let any precipitated material run through the flow-path? |
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Yes. One main feature of AP-3000 is that its flow-path is wide and short. Because of this feature, while the amount of the solution used is same with that of the other SPR devices, AP-3000 has the advantage of its flow-path not easily clogged up with non soluble materials. At the same time, a pressure sensor is installed in AP-3000, and if clogging takes place, the sensor senses it, and stops the pump to keep the solution from flowing through the flow path. If the flow-path gets clogged, the sensor stick can be replaced by a user after the measurement is taken, and any major maintenance by the vender is not needed. |
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When screening is done for analyte solutions with high concentration, non-specific binding (NSB) or aggregation of compounds often takes place, and therefore there will be a lot of false positives. Because of this reason, an SPR method seems to be unsuitable for screening of analyte solutions with high concentration. Is it true? |
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The following steps are recommended to do SPR based screening at high concentration levels.
(1)Screen all the compounds at one level of concentration.
(2)Do dose response measurements for hit compounds (i.e. 8 levels of concentration, n=2).
(3)Normalize the binding amount of compounds as the binding activity (%), using the amount of protein immobilization and the molecular weight of proteins and compounds. Those compounds saturated at the binding activity of less than 100% at the dose response are considered to be correct hit compounds (KD can be calculated.) The compounds of which the concentration dependant signal goes up and exceeds over 100% are NSB or aggregation, and should be eliminated. |
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What is the upper limit of detergent concentration? |
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The upper limit for Tween 20 is 0.05%. Consult with us for other detergents. |
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