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Plant Amino-Peptidase ELISA Kit instruction Intended use This Amino-Peptidase ELISA kit is intended Laboratory for Research use only and is not for use in diagAmino-Peptidasetic or therapeutic procedures.The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm using a spectrophotometer. In order to measure the concentration of Amino-Peptidase in the sample, this Amino-Peptidase ELISA Kit includes a set of calibration standards. The calibration standards are assayed at the same time as the samples and allow the operator to produce a standard curve of Optical Density versus Amino-Peptidase concentration. The concentration of Amino-Peptidase in the samples is then determined by comparing the O.D. of the samples to the standard curve. Sample collection and storages Can t detect the samples which contain NaN3, because NaN3 inhibits HRP activity of the horseradish peroxidase. 2. Extract as soon as possible after Specimen collection, Extracted according to the relevant literature. Cell culture supernates and plant exact fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20C or -80C. Avoid repeated freeze-thaw. Materials required but not supplied 1. Standard microplate reader(450nm) 2. Precision pipettes and Disposable pipette tips. 3. 37 ! incubator Precautions 1. Do not substitute reagents from one kit to another. Standard, conjugate and microplates are matched for optimal performance. Use only the reagents supplied by manufacturer. 2. Do not remove microplate from the storage bag until needed. Unused strips should be stored at 2-8C in their pouch with the desiccant provided. 3. Mix all reagents before using. Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25C) Materials supplied Name96 determinations48 determinationsMicroelisa stripplate12*8strips12*4stripsStandard0.3ml*6tubes0.3ml*6tubesSample Diluent6.0ml3.0mlHRP-Conjugate reagent10.0ml5.0ml20X Wash solution25ml15mlChromogen Solution A6.0ml3.0mlChromogen Solution B6.0ml3.0mlStop Solution6.0ml3.0mlClosure plate membrane22User manual11Sealed bags11Note: Standard (S0 ! S5) concentration was followed by: 0,50,100,200,400,800 U/ml Reagent preparation 20wash solution:Dilute with Distilled or deionized water 1:20. Assay procedure 1. Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate. 2. Add standard: Set Standard wells, testing sample wells. Add standard 50l to standard well. 3. Add Sample: Add testing sample 10l then add Sample Diluent 40l to testing sample well; Blank well doesn t add anyting. 4. Add 100l of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37C. 5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400l) using a squirt bottle, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels. 6. Add chromogen solution A 50l and chromogen solution B 50l to each well. Gently mix and incubate for 15 minutes at 37C. Protect from light. 7. Add 50l Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing. 8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes. Calculation of results This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve. The sensitivity by this assay is 10.0 U/ml Standard curve  Storage 2-8!. validity six months. FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC! 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