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Human soluble apoptosis related factor ligand (sFASL) ELISA kit
Human soluble apoptosis-associated factor ligand (sFASL) ELISA kit instructions Human soluble apoptosis-associated factor ligand (sFASL) ELISA kit is for research use only. Detection range: 156pg / ml -10000pg / ml Expected application: ELISA quantitative determination The content of sFASL in human serum, plasma, cell culture supernatant or other related biological fluids. Experimental principle: The microtiter plate is coated with purified antibody to make a solid phase carrier, and the specimen or standard, biotinylated anti-sFASL antibody, and HRP-labeled avidin are sequentially added to the microwell coated with anti-sFASL antibody , After thorough washing, develop color with substrate TMB. TMB is converted into blue under the catalysis of peroxidase, and into the final yellow under the action of acid. The color depth is positively correlated with sFASL in the sample. The absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm, and the sample concentration was calculated. Instructions: 1. There may be inconsistencies between the Chinese and English instructions. Please refer to the English instructions. 2. The newly opened enzyme-linked plate may contain a little water-like substance in the well. This is a normal phenomenon and will not have any impact on the experimental results. 3. Store the kit: -20 ℃ (when not in use for a long time); 2-8 ℃ (when used frequently). 4. The concentrated washing liquid will be salted out at low temperature, and it can be heated and dissolved in the water bath when diluted. Composition of human soluble apoptosis-related factor ligand (sFASL) ELISA kit and reagent preparation 1. Assay plate: one piece (96 wells). 2. Standard product (Standard): 2 bottles (lyophilized product). 3. Sample Diluent: 1 × 20ml / bottle. 4. Biotin-antibody Diluent: 1 × 10ml / bottle. 5. Horseradish peroxidase-labeled avidin dilution (HRP-avidin Diluent): 1 × 10ml / bottle. 6. Biotin-antibody: 1 × 120μl / vial (1: 100) 7. Horseradish peroxidase-labeled avidin (HRP-avidin): 1 × 120μl / vial (1: 100) 8. Substrate solution (TMB Substrate): 1 × 10 ml / bottle. 9. Wash Buffer: 1 × 20ml / bottle, each bottle is diluted 25 times with distilled water. 10. Stop Solution (Stop Solution): 1 × 10ml / bottle (2N H2SO4). Reagents and equipment not needed: 1. Standard microplate reader 2. High-speed centrifuge 3. Electric thermostatic incubator 4. Clean test tubes and Eppendof tubes 5. Series adjustable pipettes and tips, one-time testing of samples When there are many, it is best to use a multi-channel pipette. 6. Collection and storage of specimens such as distilled water and volumetric flasks: 1. Serum: whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000x g for 20 minutes. The supernatant can be detected, or the specimen can be stored at -20 ℃ or -80 ℃, but repeated freezing and thawing should be avoided. 2. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge at 1000 xg for 15 minutes at 2-8 ° C within 30 minutes after collection, or store the specimen at -20 ° C or -80 ° C, but avoid repeated freezing and thawing. 3. Cell culture supernatant or other biological specimens: centrifuge at 1000xg for 20 minutes, take the supernatant for detection, or store the specimen at -20 ℃ or -80 ℃, but avoid repeated freezing and thawing. Note: Hemolysis of specimens will affect the final test results, so hemolysis specimens should not be tested. Dilution principle of the specimen: firstly, the approximate content of the sample to be tested is determined through literature search, and the appropriate dilution factor is determined. Only when it is diluted to the range of the standard curve, the test result is accurate. Detailed records should be made during the dilution process. When calculating the concentration at the end, it was diluted "N" times, and the concentration of the specimen should be multiplied by "N". Standard dilution principle: 2 bottles, each bottle is diluted with sample diluent to 1ml before use, and left to stand for more than 10 minutes after being capped, and then inverted / rubbed repeatedly to help dissolve, its concentration is 10000pg / ml, do series After multiple dilution, dilute 10000 pg / ml, 5000 pg / ml, 2500 pg / ml, 1250 pg / ml, 625 pg / ml, 312 pg / ml, 156 pg / ml. The sample dilution is directly used as the standard concentration of 0 pg / mL. Prepare within 15 minutes before use. For example, to prepare a 5000pg / ml standard: take 0.5ml (not less than 0.5ml) of the above standard at 10000pg / ml and add it to an Eppendorf tube containing 0.5ml of sample diluent, mix well, and so on for the remaining concentrations. Dilution principle of biotin-labeled antibody: before use, dilute with biotin-labeled antibody diluent. Before dilution, prepare according to the pre-calculated total amount required for each experiment (100μl per well). 0.1- 0.2ml. For example, 10 μl of biotin-labeled antibody plus 990 μl of biotin-labeled antibody dilution is prepared, mixed gently, and prepared within one hour before use. Dilution principle of horseradish peroxidase-labeled avidin: before use, dilute with horseradish peroxidase-labeled avidin diluent, and prepare according to the pre-calculated total amount required for each experiment before dilution (Each 100μl), 0.1-0.2ml should be prepared in actual preparation. For example, 10 μl horseradish peroxidase-labeled avidin plus 990 μl horseradish peroxidase-labeled avidin dilution is prepared, mix gently, and prepare within one hour before use. Operation steps: Before starting the experiment, please configure all the reagents in advance. When the reagents or samples are diluted, they should be mixed evenly. Try to avoid foaming when mixing. A standard curve should be made for each test. If the sample concentration is too high, dilute with sample diluent to make the sample meet the detection range of the kit. 1. Add sample: set blank hole, standard hole and sample hole to be tested respectively. Add 100μl of sample diluent to blank wells, and add 100μl of standard or test sample to the remaining wells. Be careful not to have air bubbles. Add samples to the bottom of the wells of the microtiter plate. Try not to touch the walls of the wells. The target plate is covered with a cover or film and reacted at 37 ° C for 120 minutes. To ensure the validity of the experimental results, please use a new standard solution for each experiment. 2. Discard the liquid and spin dry without washing. Add 100 μl of biotinylated antibody working solution to each well (the ratio of 1 μl of biotinylated antibody plus 99 μl of biotinylated antibody dilution is prepared, mix gently and prepare within one hour before use), 37 ° C, 60 minutes. 3. After incubating for 60 minutes, discard the liquid in the wells, spin dry, wash the plate 3 times, soak for 1-2 minutes each time, 350μl / well, spin dry. 4. Add 100 μl of horseradish peroxidase-labeled avidin working solution (with biotin-labeled antibody working solution) to each well at 37 ° C for 60 minutes. 5. After incubating for 60 minutes, discard the liquid in the well, spin dry, wash the plate 5 times, soak for 1-2 minutes each time, 350μl / well, spin dry. 6. Add 90μl of substrate solution to each well in sequence, and develop color at 37 ° C in the dark (within 30 minutes, at this time, the first 3-4 wells of the standard product have a visible blue gradient before the 3-4 wells, and the gradient of the back 3-4 wells is not obvious. , You can terminate). 7. Add 50μl of stop solution to each well in sequence to stop the reaction (at this time, the blue color turns to yellow). The order of adding the stop solution should be the same as that of the substrate solution. In order to ensure the accuracy of the experimental results, the termination solution should be added as soon as possible after the substrate reaction time expires. 8. Measure the optical density (OD value) of each well in sequence using an enzyme-linked instrument at a wavelength of 450 nm. Test within 15 minutes after adding stop solution. Note: 1. When using the kit for the first time, the user should centrifuge various reagent tubes for a few minutes so that the reagents are concentrated to the bottom of the tube. 2. Leave one well for each experiment as a blank zero-adjusting well. No reagents are added to this well, only the substrate solution and 2N H2SO4 are added at the end. Use this hole to adjust the OD value to zero when measuring. 3. To prevent the sample from evaporating, place the reaction plate in a closed box covered with a damp cloth during the test, and add a cover or film to the enzyme label plate. 4. Store unused microplates or reagents at 2-8 ° C. Standards, biotin-labeled antibody working solution, horseradish peroxidase-labeled avidin working solution, please use according to the required amount. Do not reuse diluted standard, biotin-labeled antibody working solution, or horseradish peroxidase-labeled avidin working solution. 5. It is recommended to set double-hole measurement when testing samples to ensure the accuracy of the test results. Washing method: automatic washing: if there is an automatic washing machine, it should be used in the formal experiment after being used skillfully. Manual plate washing method: Aspirate (do not touch the wall) or shake off the liquid in the microplate; put a few layers of absorbent paper on the experimental table, and force the microplate down several times; the recommended wash buffer is at least 0.3 ml is injected into the hole and soaked for 1-2 minutes. Repeat this process several times as needed. Calculation: Taking the concentration of the standard as the abscissa (logarithmic coordinate) and the OD value as the ordinate (ordinary coordinate), draw a standard curve on semi-logarithmic coordinate paper, and find the corresponding curve from the standard curve according to the OD value of the sample Concentration; multiply by the dilution factor; or calculate the linear regression equation of the standard curve using the concentration of the standard and the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and multiply by the dilution factor, which is the actual sample concentration. Matters needing attention: 1. When mixing protein solution, it should be as gentle as possible to avoid foaming. 2. The washing process is very important, inadequate washing is easy to cause false positives. 3. It is best to control the sampling time within 5 minutes. If there are many specimens, it is recommended to use a volley gun to add samples. 4. Please make a standard curve at the same time of each measurement, it is best to make a double hole. 5. If the content of the substance to be tested in the specimen is too high, please dilute it and then determine it. When calculating, please multiply by the dilution factor. 6. When preparing standard products and testing solution working fluid, please prepare with corresponding diluent, not to be confused. 7. Please keep the substrate away from light. 8. Do not replace the reagent specificity in the kit with reagents from other manufacturers: this kit can detect natural or recombinant sFASL at the same time, and does not cross-react with other related proteins. Validity: 6 months