Peroxisome Proliferator Activated Receptor Alpha (PPARa) BioAssay(TM) ELISA Kit (Human)

The Human Peroxisome Activated Receptor Alpha (PPARa) ELISA Kit is a sandwich enzyme immunoassay for the in vitro quantitative measurement of PPARa in human tissue homogenates and other biological fluids. Detection Range: 0.156-10ng/ml, Sensitivity: <0.052ng/ml, Precision: 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 PPARa. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to PPARa. 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 PPARa, 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 PPARa in the sample is then determined by comparing the O.D. of the sample to the standard curve. Kit Components: *027378A: Microtiter Plate, 1x96 wells, Pre-coated, ready to use, *027378B: Standard, 2x1vial , 027378C: Standard Diluent, 1x20ml, *027378D: Detection Reagent A, 1x120ul , *027378E: Detection Reagent B, 1x120ul , 027378F: Assay Diluent A, 1x12ml, 027378G: Assay Diluent B, 1x12ml, 027378H: TMB Substrate, 1x9ml, 027378K: Stop Solution, 1x6ml, 027378L: Wash Buffer, 30X, 1x20ml , Storage and Stability: Store *027378A, *027378B, *027378D and *027378E 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 Solution, Sample Preparation and Storage: Tissue Homogenates:, The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissues were rinsed thoroughly in ice-cold PBS (0.02M pH 7.0-7.2) to remove excess blood and weighed before homogenization. The tissues were minced to small pieces and homogenized in 5-10ml of PBS with a glass homogenizer on ice (Micro Tissue Grinders works too). The resulting suspension was sonicated with an ultrasonic cell disrupter or subjected to two freeze-thaw cycles to further break the cell membranes. After that, the homogenates were centrifuged for 5 minutes at 2500×g. The supernatant was removed and assayed immediately or aliquoted and stored at -20°C or lower. 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., Note:, 1. Samples to be used within 5 days may be stored at 4°C, otherwise samples must be stored at -20°C (?1 month) or -80°C (?2 months) to avoid loss of bioactivity and contamination., 2. Sample hemolysis will influence the results so hemolyzed specimens should not be used., 3. When performing the assay, bring samples to RT. 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.,