The Mouse Toll-Like Receptor 5 （TLR5） ELISA kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of TLR5 in mouse tissue homogenates，cell lysates，culture supernatants and other biological fluids.Detection Range：0.312 – 20ng/ml.Sensitivity：<0.133ng/mlPrecision：Intra-Assay： CV<10%Inter-Assay： CV<12%Test Principle：The microtiter plate provided in this kit has been pre-coated with an antibody specific to TLR5. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to TLR5. Next，Avidin conjugated to Horseradish Peroxidase （HRP） is added to each microplate well and incubated. After TMB substrate solution is added，only those wells that contain TLR5，biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ± 10nm. The concentration of TLR5 in the sample is then determined by comparing the O.D. of the sample to the standard curve.Kit Components：*366746A： Microtiter Plate，1x96 wells，Pre-coated; ready to use.*366746B： Standard，2x1vial 366746C： Standard Diluent，1x20ml*366746D： Detection Reagent A，1x120ul *366746E： Detection Reagent B，1x120ul 366746F： Assay Diluent A，1x12ml366746G： Assay Diluent B，1x12ml366746H： TMB Substrate，1x9ml366746K： Stop Solution，1x6ml366746L： Wash Buffer，30X，1x20ml Storage and Stability：Store *366746A，*366746B，*366746D and *366746E at -20°C. Store all the other components at 4°C. Unused kit is stable for 6 months. Once kit components are opened，it is highly recommended to use remaining reagents within 1 month provided this is within the expiration date of the kit. For maximum recovery of product，centrifuge the original vials after thawing and prior to removing the cap.Materials Required But Not Supplied：1. Microplate reader with 450 ± 10nm filter.2. Precision single or multi-channel pipettes and disposable tips.3. Eppendorf Tubes for diluting samples.4. Deionized or distilled water.5. Absorbent paper for blotting the microtiter plate.6. Container for Wash SolutionSample Preparation and Storage：Cell Lysates： Cells must be lysed before assaying according to the following directions. 1. Adherent cells should be detached with trypsin and then collected by centrifugation at 1000xg for 5 minutes （suspension cells can be collected by centrifugation directly）. 2. Wash cells three times in cold PBS. 3. Resuspend cells in PBS （1×） at a concentration of 10e7 cells per ml. If it is necessary，subject the cells to ultrasonication until the solution is clarified.4. Centrifuge at 1500×g for 10 minutes at 2-8°C to remove cellular debris. Assay immediately or aliquot and store at at -20°C or lower.Tissue Homogenates：The preparation of tissue homogenates will vary depending upon tissue type.1. Rinse tissue thoroughly in ice-cold PBS （0.02M pH 7.0-7.2） to remove excess blood and weigh before homogenization. 2. Mince the tissue to small pieces and homogenize in fresh lysis buffer （1ml lysis buffer per 20-50mg tissue sample） using a glass homogenizer on ice （micro tissue grinders may also be used）. Note： Choice of lysis buffer depends on the sub-cellular localization of the target protein.3. Subject the resulting suspension to sonication using an ultrasonic cell disrupter until a clarified mixture is obtained.4. Centrifuge the homogenate for 5 minutes at 10,000 x g. Collect the supernatant and assay immediately or aliquot and store at -20°C or lower.Cell Culture Supernatants and Other Biological Fluids：Centrifuge samples for 20 minutes at 1000×g. Remove particulates and assay immediately or store samples in aliquots at -20°C or -80°C. Avoid repeated freeze/thaw cycles.Assay Procedure Summary：1. Prepare all reagents，samples and standards.2. Add 100ul standard or sample to each well. Incubate 2 hours at 37°C.3. Aspirate and add 100ul prepared Detection Reagent A. Incubate 1 hour at 37°C.4. Aspirate and wash 3 times.5. Add 100ul prepared Detection Reagent B. Incubate 30 minutes at 37°C.6. Aspirate and wash 5 times.7. Add 90ul TMB Substrate. Incubate 15-25 minutes at 37°C.8. Add 50ul Stop Solution. Read at 450nm immediately.
